Modified factor ix polypeptides and uses thereof

ABSTRACT

Nucleic acid molecules encoding modified factor IX (FIX) polypeptides and uses thereof are provided. Such modified FIX polypeptides include FIXa and other forms of FIX. Among the modified FIX polypeptides provided are those that have altered activities, typically altered procoagulant activity, including increased procoagulant activities. Hence, such modified polypeptides are therapeutics.

RELATED APPLICATIONS

This application is a divisional of allowed U.S. patent application Ser.No. 14/267,754, entitled “MODIFIED FACTOR IX POLYPEPTIDES AND USESTHEREOF,” filed on May 1, 2014, to Edwin Madison, Christopher Thanos andGrant Ellsworth Blouse, which is a continuation of U.S. patentapplication Ser. No. 13/373,118, entitled “MODIFIED FACTOR IXPOLYPEPTIDES AND USES THEREOF,” filed on Nov. 3, 2011, now U.S. Pat. No.8,778,870, issued Jul. 15, 2014, to Edwin Madison, Christopher Thanosand Grant Ellsworth Blouse, which claims the benefit of priority to U.S.Provisional Application Ser. No. 61/456,298, filed on Nov. 3, 2010,entitled “MODIFIED FACTOR IX POLYPEPTIDES AND USES THEREOF,” to EdwinMadison, Christopher Thanos and Grant Ellsworth Blouse.

This application also is related to International PCT Application No.PCT/US11/59233, filed on Nov. 3, 2011, published as International PCTApplication No. WO 2012/061654, and entitled “MODIFIED FACTOR IXPOLYPEPTIDES AND USES THEREOF,” which also claims priority to U.S.Provisional Application Ser. No. 61/456,298.

The subject matter of each of the above-referenced applications isincorporated by reference in its entirety.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED ELECTRONICALLY

An electronic version of the Sequence Listing is filed herewith, thecontents of which are incorporated by reference in their entirety. Theelectronic file was created on Mar. 24, 2016, is 1.07 megabytes in size,and titled 4918Cseq001.txt.

FIELD OF INVENTION

Provided are modified FIX polypeptides. The FIX polypeptides aremodified to exhibit improved properties, such as increased coagulantactivity compared to unmodified FIX polypeptides. Also provided arenucleic acid molecules encoding these polypeptides, and methods of usingthe modified FIX polypeptides.

BACKGROUND OF THE INVENTION

Recombinantly produced Factor IX (FIX) polypeptides have been approvedfor treatment of hemophilia, in particular hemophilia B. Also oftherapeutic interest are FIX polypeptides that exhibit anticoagulantactivities useful in the treatment of thrombolytic diseases. Hence, FIX,like other coagulation factors, are important therapeutic agents forprocoagulant and anticoagulation therapies. There is a need for FIXpolypeptides for therapeutic use. Therefore, among the objects herein,it is an object to provide modified FIX polypeptides that are designedto have improved therapeutic properties.

SUMMARY

Provided are modified FIX polypeptides. The modified FIX polypeptidesprovided have improved procoagulant therapeutic properties compared toan unmodified FIX polypeptide. For example, among the modified FIXpolypeptides provided herein are those that exhibit increased coagulantactivity, increased catalytic activity, increased resistance to AT-III,heparin and/or the AT-III/heparin complex, and/or improvedpharmacokinetic properties, such as i) decreased clearance, ii) altered(e.g. increased or decreased) volume of distribution, iii) enhanced invivo recovery, iv) enhanced total protein exposure in vivo (i.e., AUC),v) increased serum half-life (α-, β-, and/or γ-phase), and/or vi)increased mean resonance time (MRT). In some examples, the improvedpharmacokinetic properties are a result of increased glycosylationand/or decreased binding to the low-density lipoprotein receptor-relatedprotein (LRP). Also provided are nucleic acids encoding the modified FIXpolypeptides and methods of using the modified FIX polypeptides, such asfor treatment of bleeding disorders.

Provided herein are modified FIX polypeptides containing an amino acidreplacement in an unmodified FIX polypeptide, wherein the amino acidreplacement can be one or more of replacement of tyrosine (Y) at aminoacid residue R318 (R318Y), R318E, R318F, R318W, R318D, R3181, R318K,R318L, R318M, R318S, R318V, S61A, S61C, S61D, S61E, S61F, S61G, S61I,S61K, S61L, S61P, S61R, S61V, S61W, S61Y, D64A, D64C, D64F, D64H, D64I,D64L, D64M, D64P, D64R, D64S, D64T, D64W, Y155F, Y155L, N157D, N157E,N157F, N157I, N157K, N157L, N157M, N157R, N157V, N157W, N157Y, S158A,S158D, S158E, S158F, S158G, S158I, S158K, S158L, S158M, S158R, S158V,S158W, S158Y, N167D, N167Q, N167E, N167F, N167G, N167H, N167I, N167K,N167L, N167M, N167P, N167R, N167V, N167W, N167Y, T169A, T169D, T169E,T169F, T169G, T169I, T169K, T169L, T169M, T169P, T169R, T169S, T169V,T169W, T169Y, T172A, T172D, T172E, T172F, T172G, T172I, T172K, T172L,T172M, T172P, T172R, T172S, T172V, T172W, T172Y, D203M, D203Y, D203F,D203H, D203I, D203K, D203L, D203R, D203V, D203W, A204M, A204Y, A204F,A204I, A204W, E239S, E239R, E239K, E239D, E239F, E239I, E239L, E239M,E239T, E239V, E239W, E239Y, H257F, H257E, H257D, H257I, H257K, H257L,H257M, H257Q, H257R, H257V, H257W, R312Y, R312L, R312C, R312D, R312E,R312F, R312I, R312K, R312M, R312P, R312S, R312T, R312V, R312W, K316M,K316D, K316F, K316H, K316I, K316L, K316R, K316V, K316W, K316Y, F342I,F342D, F342E, F342K, F342L, F342M, F342S, F342T, F342V, F342W, F342Y,T343R, T343E, T343D, T343F, T343I, T343K, T343L, T343M, T343S, T343V,T343W, T343Y, N346Y, N346E, N346F, N346H, N346I, N346K, N346L, N346M,N346Q, N346R, N346V, N346W, K400E, K400C, K400D, K400F, K400G, K400L,K400M, K400P, K400S, K400T, K400V, K400Y, R403D, R403F, R403I, R403K,R403L, R403M, R403S, R403V, R403Y, E410D, E410S, E410A, E410F, E410G,E410I, E410K, E410L, E410M, E410P, E410R, E410T, E410V, E410W, E410Y,T412A, T412V, T412C, T412D, T412E, T412F, T412G, T412I, T412M, T412P,T412W or T412Y in a mature FIX polypeptide having a sequence set forthin SEQ ID NO:3, or the same replacement at a corresponding amino acidresidue in an unmodified FIX polypeptide, wherein corresponding aminoacid residues are identified by alignment of the unmodified FIXpolypeptide with the polypeptide of SEQ ID NO:3; and provided that themodified FIX polypeptide does not contain the modificationsF342I/T343R/Y345T. In particular, provided herein are modified FIXpolypeptides containing amino acid replacements R318Y/R338E/R403E/E410N,R318Y/R338E/T343R/R403E/E410N, R318Y/R338E/T343R/E410N,Y155F/R318Y/R338E/T343R/R403E,Y155F/K228N/K247N/N249S/R318Y/R338E/T343R/R403E/E410N,Y155F/K247N/N249S/R318Y/R338E/T343R/R403E,K247N/N249S/R318Y/R338E/T343R/R403E, R318Y/R338E/T343R,Y155F/K247N/N249S/R318Y/R338E/T343R,K228N/R318Y/R338E/T343R/R403E/E410N,K228N/K247N/N249S/R318Y/R338E/T343R/R403E,R318Y/R338E/T343R/R403E/E410S, Y155F/K247N/N249S/R318Y/R338E,K247N/N249S/R318Y/R338E/T343R, R318Y/T343R/E410N,Y155F/R318Y/R338E/R403E, Y155F/R338E/T343R/R403E/E410N,Y155F/K247N/N249S/R338E/R403E/E410N, K247N/N249S/R338E/T343R/R403E/E410Nor R338E/T343R/E410N.

Among the modified FIX polypeptides provided herein are those containingtwo amino acid replacements in unmodified FIX polypeptide, wherein thefirst amino acid replacement is at a position corresponding to aposition selected from among 53, 61, 64, 85, 103, 104, 105, 106, 108,155, 158, 159, 167, 169, 172, 179, 202, 203, 204, 205, 228, 239, 241,243, 247, 249, 251, 257, 259, 260, 262, 265, 284, 293, 312, 314, 315,316, 317, 318, 319, 321, 333, 338, 343, 346, 345, 392, 394, 400, 403,410, 412 and 413 in a mature FIX polypeptide having a sequence set forthin SEQ ID NO:3, and the second amino acid replacement is at a positioncorresponding to a position selected from among 5, 53, 61, 64, 85, 155,158, 159, 167, 239, 260, 284, 293, 312, 318, 333, 338, 346, 400, 403,410, 412 and 413 in a mature FIX polypeptide having a sequence set forthin SEQ ID NO:3.

In some examples, the first or the second amino acid replacement is areplacement with an amino acid residue selected from among alanine (Ala,A); arginine (Arg, R); asparagine (Asn, N); aspartic acid (Asp, D);cysteine (Cys, C); glutamic acid (Glu, E); glutamine (Gln, Q); glycine(Gly, G); histidine (His, H); isoleucine (Ile, I); leucine (Leu, L);lysine (Lys, K); methionine (Met, M); phenylalanine (Phe, F); proline(Pro, P); serine (Ser, S); threonine (Thr, T); tryptophan (Trp, W);tyrosine (Tyr, Y); and valine (Val, V), providing the replacing aminoacid is not the same as the amino acid it replaces. In particularexamples, the first amino acid replacement is a replacement with anamino acid residue selected from among alanine; asparagine; asparticacid, glutamic acid; glutamine; histidine; isoleucine; leucine; lysine;methionine; phenylalanine; serine; threonine; tyrosine and valine. Forexample, exemplary amino acid replacements include S53A, S61A, D64A,D64N, D85N, A103N, D104N, N105S, K106S, K106N, V108S, Y155F, Y155H,Y155Q, S158A, S158D, S158E, T159A, N167D, N167Q, T169A, T172A, T179A,V202M, V202Y, D203M, D203Y, A204M, A204Y, K228N, E239A, E239N, E239S,E239R, E239K, T241N, H243S, K247N, N249S, 1251S, H257F, H257E, H257F,H257Y, H257S, Y259S, N260S, A262S, K265T, Y284N, K293E, K293A, R312Q,R312A, R312Y, R312L, F314N, H315S, K316S, K316N, K316A, K316E, K316S,K316M, G317N, R318A, R318E, R318Y, R318N, S319N, A320S, L321S, R333A,R333E, R333S, R338A, R338E, R338L, T343R, T343E, T343Q, F342I, Y345A,Y345T, N346D, N346Y, K392N, K394S, K400A, K400E, R403A, R403E, E410Q,E410N, E410D, E410S, E410A, T412A, T412V or K413N. Other exemplary aminoacid replacements are conservative amino acid replacements.

In some instances, the second amino acid replacement is a replacementwith an amino acid residue selected from among alanine; arginine;asparagine; aspartic acid; glutamic acid; glutamine; histidine; leucine;lysine; phenylalanine; serine; threonine; tyrosine; or valine. Forexample, exemplary amino acid replacements include K5A, S53A, S61A,D64A, D64N, D85N, Y155F, Y155H, Y155Q, S158A, S158D, S158E, T159A,N167D, N167Q, E239A, E239N, E239S, E239R, E239K, N260S, Y284N, K293E,K293A, R312Q, R312A, R312Y, R312L, R318A, R318E, R318Y, R318N, R333A,R333E, R333S, R338A, R338E, R338L, N346D, N346Y, K400A, K400E, R403A,R403E, E410Q, E410N, E410D, E410S, E410A, T412A, T412V or K413N. Otherexemplary amino acid replacements are conservative amino acidreplacements.

In particular examples, the first amino acid replacement is at aposition corresponding to a position selected from among 155, 247, 249,318, 338, 403 and 410, such as, for example, Y155F, K247N, N249S, R318Y,R338E, R403E and E410N. In further examples, the second amino acidreplacement is at a position corresponding to a position selected fromamong 155, 247, 249, 318, 338, 403 and 410, such as, for example, Y155F,K247N, N249S, R318Y, R338E, R403E and E410N.

Among the modified FIX polypeptides provided herein are those containingamino acid replacements selected from among amino acid replacementscorresponding to K400E/R403E, R318E/R403E, R318Y/E410N, K228N/R318Y,Y155F/K228N, Y155F/1251S, Y155F/N346D, Y155F/N260S, R338E/T343R,E410N/T412A, E410N/T412V, R318Y/R338E, D85N/K228N, D85N/1251S,K400A/R403A, R338A/R403A, R338E/R403E, K293A/R403A, K293E/R403E,R318A/R403A, R338E/E410N, K228N/E410N, K228N/R338E, K228N/R338A andR403E/E410N.

In some examples, the modified FIX polypeptides contain one or morefurther amino acid replacements, such as one or more at a positionselected from among 53, 61, 64, 85, 103, 104, 105, 106, 108, 155, 158,159, 167, 169, 172, 179, 202, 203, 204, 205, 228, 239, 241, 243, 247,249, 251, 257, 259, 260, 262, 265, 284, 293, 312, 314, 315, 316, 317,318, 319, 321, 333, 338, 343, 346, 345, 392, 394, 400, 403, 410, 412 and413 in a mature FIX polypeptide having a sequence set forth in SEQ IDNO:3. For example, the modified FIX polypeptides can contain a furtheramino acid replacement selected from among Y5A, S53A, S61A, D64A, D64N,D85N, A103N, D104N, N105S, K106S, K106N, V108S, Y155F, Y155H, Y155Q,S158A, S158D, S158E, T159A, N167D, N167Q, T169A, T172A, T179A, V202M,V202Y, D203M, D203Y, A204M, A204Y, K228N, E239A, E239N, E239S, E239R,E239K, T241N, H243S, K247N, N249S, I251S, H257F, H257E, H257F, H257Y,H257S, Y259S, N260S, A262S, K265T, Y284N, K293E, K293A, R312Q, R312A,R312Y, R312L, F314N, H315S, K316S, K316N, K316A, K316E, K316S, K316M,G317N, R318A, R318E, R318Y, R318N, S319N, A320S, L321S, R333A, R333E,R333S, R338A, R338E, R338L, T343R, T343E, T343Q, F342I, Y345A, Y345T,N346D, N346Y, K392N, K394S, K400A, K400E, R403A, R403E, E410Q, E410N,E410D, E410S, E410A, T412A, T412V and K413N, or a conservative aminoacid replacement.

In some examples, the modified FIX polypeptides provided herein containamino acid replacements selected from among amino acid replacementscorresponding to R318Y/R338E/R403E, D203N/F205T/R318Y,R318Y/R338E/E410N, K228N/R318Y/E410N, R318Y/R403E/E410N,R318Y/R338E/R403E/E410N, D203N/F205T/R318Y/E410N,A103N/N105S/R318Y/R338E/R403E/E410N,D104N/K106S/R318Y/R338E/R403E/E410N, K228N/R318Y/R338E/R403E/E410N,I251S/R318Y/R338E/R403E/E410N,D104N/K106S/I251S/R318Y/R338E/R403E/E410N,D104N/K106S/R318Y/E410N/R338E, 1251S/R318Y/E410N/R338E,D104N/K106S/I251S/R318Y/E410N/R338E, A103N/N105S/Y155F,D104N/K106S/Y155F, Y155F/K247N/N249S,A103N/N105S/K247N/N249S/R318Y/R338E/R403E/E410N,D104N/K106S/K247N/N249S/R318Y/R338E/R403E/E410N,K228N/K247N/N249S/R318Y/R338E/R403E/E410N,A103N/N105S/Y155F/R318Y/R338E/R403E/E410N,D104N/K106S/Y155F/R318Y/R338E/R403E/E410N,Y155F/K228N/R318Y/R338E/R403E/E410N,Y155F/I251S/R318Y/R338E/R403E/E410N,Y155F/K247N/N249S/R318Y/R338E/R403E/E410N,K247N/N249S/R318Y/R338E/R403E/E410N, Y155F/R318Y/R338E/R403E/E410N,K247N/N249S/R318Y/R338E/E240N, Y155F/R318Y/R338E/E410N,Y155F/K247N/N249S/R318Y/R338E/E410N,D104N/K106S/Y155F/K228N/K247N/N249S, D104N/K106S/Y155F/K247N/N249S,D104N/K106S/Y155F/K228N, Y155F/K228N/K247N/N249S,R318Y/R338E/R403E/E410S, R318Y/R338E/R403E/E410N/T412V,R318Y/R338E/R403E/E410N/T412A, R318Y/R338E/R403E/T412A,R318Y/R338E/E410S, R318Y/R338E/T412A, R318Y/R338E/E410N/T412V,D85N/K228N/R318Y/R338E/R403E/E410N, N260S/R318Y/R338E/R403E/E410N,R318Y/R338E/N346D/R403E/E410N, Y155F/R318Y/R338E/N346D/R403E/E410N,Y155F/N260S/N346D, K247N/N249S/N260S/R318Y/R338E/R403E/E410N,D104N/K106S/N260S/R318Y/R338E/R403E/E410N,Y155F/N260S/R318Y/R338E/R403E/E410N, R318Y/R338E/T343R/R403E/E410N,D104N/K106S/Y155F/N260S, Y155F/K247N/N249S/N260S,D104N/K106S/Y155F/K247N/N249S/N260S, D104N/K106S/Y155F/K228N,D104N/K106S/Y155F/K247N/N249S, D85N/D203N/F205T, D85N/D104N/K106S/1251S,K293A/R338A/R403A, K293E/R338E/R403E, R338E/R403E/E410N,D203N/F205T/K228N, D203N/F205T/E410N, D203N/F205T/R338E,D203N/F205T/R338A, D203N/F205T/R338E/R403E, K228N/R338E/R403E,K247N/N249S/N260S, D104N/K106S/N260S, K228N/K247N/N249S/D104N/K106S,A103N/N105S/K228N, D104N/K106S/K228N, A103N/N105S/I2515,D104N/K106S/I251S, A103N/N105S/K247N/N249S, D104N/K106S/K247N/N249S,K228N/K247N/N249S, D104N/K106S/K228N/K247N/N249S, K247N/N249S/N260S,D104N/K106S/N260S, Y259F/K265T/Y345T and D104N/K106S/K247N/N249S/N260S.

Also provided herein are modified FIX polypeptides containing amodification in an unmodified FIX polypeptide, wherein the modificationis selected from among modifications corresponding to amino acidreplacements S61A, D64A, Y155F, N157D, S158A, S158D, S158E, N167D,N167Q, T169A, T172A, D203M, D203Y, A204M, A204Y, E239S, E239R, E239K,H257F, H257E, R312Y, R312L, K316M, R318E, R318Y, T343R, T343E, F342I,N346Y, K400E, E410D, E410S, E410A, T412A and T412V in a mature FIXpolypeptide having a sequence set forth in SEQ ID NO:3. In someexamples, the modified FIX polypeptide contains two or more of the aminoacid replacements.

In particular instances, the modified FIX polypeptide contains themutation Y155F. For example, provided are modified FIX polypeptides thatcontain Y155F and a modification at an amino acid position selected fromamong positions corresponding to 247, 249, 338, 403 and 410 of a matureFIX polypeptide having a sequence set forth in SEQ ID NO:3. In oneexample, the modified FIX contains Y155F/K247N/N249S. In furtherinstances, the modified FIX polypeptide contains the mutation R318Y. Forexample, provided are modified FIX polypeptides containing R318Y and amodification at an amino acid position selected from positionscorresponding to 338, 403 and 410 of a mature FIX polypeptide having asequence set forth in SEQ ID NO:3, such as, for example, R338E, R403E orE410N.

In some examples, the modified FIX polypeptides contain one or morefurther modifications at an amino acid position selected from amongpositions corresponding to 5, 53, 61, 64, 85, 103, 104, 105, 106, 108,148, 155, 157, 158, 159, 167, 169, 172, 179, 202, 202, 203, 204, 205,228, 239, 241, 243, 247, 249, 251, 257, 259, 260, 262, 265, 284, 293,312, 314, 315, 316, 317, 318, 319, 320, 321, 333, 338, 343, 345, 346,392, 394, 400, 403, 410, 412 and 413 of a mature FIX polypeptide havinga sequence set forth in SEQ ID NO:3. Exemplary modification(s) areselected from among modifications corresponding to amino acidreplacements K5A, S53A, S61A, D64A, D64N, D85N, A103N, D104N, N105S,N105T, K106N, K106N, K106T, V108S, V108T, T148A, Y155F, Y155H, N157D,N157Q, S158A, S158D, S158E, T159A, N167D, N167Q, T169A, T172A, T179A,V202M, V202Y, D203M, D203Y, D203N, A204M, A204Y, F205S, F205T, K228N,E239N, T241N, E239S, E239A, E239R, E239K, H243S, H243T, K247N, N249S,N249T, 1251S, I251T, H257F, H257Y, H257E, H257S, N260S, A262S, A262T,Y284N, K293E, K293A, R312Q, R312A, R312Y, R312L, F314N, H315S, K316S,K316T, K316M, G317N, R318E, R318Y, R318N, R318A, S319N, A320S, L321N,L321S, L321T, R333A, R333E, R338A, R338E, T343R, T343E, T343Q, F342I,Y345A, Y345T, N346D, N346T, K392N, K394S, K394T, K400A, K400E, R403A,R403E, E410Q, E410S, E410N, E410A, E410D, T412V, T412A and K413N.

Thus, provided herein are modified FIX polypeptides containingmodifications selected from among modifications corresponding to aminoacid replacements K400E/R403E, R318E/R403E, R318Y/E410N,R318Y/R338E/R403E, D203N/F205T/R318Y, K228N/R318Y, R318Y/R338E/E410N,K228N/R318Y/E410N, R318Y/R403E/E410N, R318Y/R338E/R403E/E410N,D203N/F205T/R318Y/E410N, A103N/N105S/R318Y/R338E/R403E/E410N,D104N/K106S/R318Y/R338E/R403E/E410N, K228N/R318Y/R338E/R403E/E410N,12515/R318Y/R338E/R403E/E410N,D104N/K106S/I251S/R318Y/R338E/R403E/E410N,D104N/K106S/R318Y/E410N/R338E, I251S/R318Y/E410N/R338E,D104N/K106S/I251S/R318Y/E410N/R338E, A103N/N105S/Y155F,D104N/K106S/Y155F, Y155F/K228N, Y155F/I251S, Y155F/K247N/N249S,A103N/N105S/K247N/N249S/R318Y/R338E/R403E/E410N,D104N/K106S/K247N/N249S/R318Y/R338E/R403E/E410N,K228N/K247N/N249S/R318Y/R338E/R403E/E410N,A103N/N105S/Y155F/R318Y/R338E/R403E/E410N,D104N/K106S/Y155F/R318Y/R338E/R403E/E410N,Y155F/K228N/R318Y/R338E/R403E/E410N,Y155F/I251S/R318Y/R338E/R403E/E410N,Y155F/K247N/N249S/R318Y/R338E/R403E/E410N,K247N/N249S/R318Y/R338E/R403E/E410N, Y155F/R318Y/R338E/R403E/E410N,K247N/N249S/R318Y/R338E/E240N, Y155F/R318Y/R338E/E410N,Y155F/K247N/N249S/R318Y/R338E/E410N,D104N/K106S/Y155F/K228N/K247N/N249S, D104N/K106S/Y155F/K247N/N249S,D104N/K106S/Y155F/K228N, Y155F/K228N/K247N/N249S,R318Y/R338E/R403E/E410S, R318Y/R338E/R403E/E410N/T412V,R318Y/R338E/R403E/E410N/T412A, R318Y/R338E/R403E/T412A,R318Y/R338E/E410S, R318Y/R338E/T412A, R318Y/R338E/E410N/T412V,D85N/K228N/R318Y/R338E/R403E/E410N, N260S/R318Y/R338E/R403E/E410N,R318Y/R338E/N346D/R403E/E410N, Y155F/N346D,Y155F/R318Y/R338E/N346D/R403E/E410N, Y155F/N260S, Y155F/N260S/N346D,K247N/N249S/N260S/R318Y/R338E/R403E/E410N,D104N/K106S/N260S/R318Y/R338E/R403E/E410N,Y155F/N260S/R318Y/R338E/R403E/E410N, R318Y/R338E/T343R/R403E/E410N,D104N/K106S/Y155F/N260S, Y155F/K247N/N249S/N260S, R338E/T343R andD104N/K106S/Y155F/K247N/N249S/N260S, D104N/K106S/Y155F/K228N,D104N/K106S/Y155F/K247N/N249S, T343R/Y345T, E410N/T412A, R410N/T412V andR318Y/R338E. In particular examples, the modified FIX polypeptidescontain modifications corresponding to amino acid replacementsR318Y/R338E/R403E/E410N or Y155F/K247N/N249S/R318Y/R338E/R403E/E410N.

In some instances, the unmodified FIX polypeptide contains a sequence ofamino acids set forth in any of SEQ ID NOS: 2, 3, 20 or 325, or is aspecies variant thereof, or a variant having at least 60% sequenceidentity with the FIX of any of SEQ ID NOS: 2, 3, 20 or 325, or is anactive fragment of a FIX polypeptide that comprises a sequence of aminoacids set forth in any SEQ ID NOS: 2, 3, 20 or 325. For example, thespecies variant can have a sequence of amino acids set forth in any ofSEQ ID NOS: 4-18. In other examples, the variant having at least 60%sequence identity with the FIX of any of SEQ ID NOS: 2, 3, 20 or 325,has a sequence of amino acids set forth in any of SEQ ID NOS: 75-272. Infurther examples, the modified FIX polypeptide is an active fragment ofan unmodified FIX polypeptide; and the modified FIX polypeptide containsthe modification(s).

Any of the modified FIX polypeptides provided herein of can contain oneor more modifications that introduces and/or eliminates one or moreglycosylation sites compared to the unmodified FIX polypeptide. In someexamples, the glycosylation sites are selected from among, N-, O- andS-glycosylation sites. In one example, one or more N-glycosylation sitesare introduced compared to the unmodified FIX polypeptide. In someexamples, the N-glycosylation site is introduced at an amino acidpositions corresponding to positions selected from among Y1, S3, G4, K5,L6, E7, F9, V10, Q11, G12, L14, E15, R16, M19, E20, K22, S24, F25, E26,E27, A28, R29, E30, V31, F32, E33, T35, E36, R37, T39, E40, F41, W42,K43, Q44, Y45, V46, D47, G48, D49, Q50, E52, S53, N54, L57, N58, G59,S61, K63, D65, 166, N67, S68, Y69, E70, W72, P74, F77, G79, K80, N81,E83, L84, D85, V86, T87, N89, 190, K91, N92, R94, K100, N101, S102,A103, D104, N105, K106, V108, S110, E113, G114, R116, E119, N120, Q121,K122, S123, E125, P126, V128, P129, F130, R134, V135, S136, S138, Q139,T140, S141, K142, A146, E147, A148, V149, F150, P151, D152, V153, D154,Y155, V156, S158, T159, E160, A161, E162, T163, 1164, L165, D166, 1168,T169, Q170, S171, T172, Q173, S174, F175, N176, D177, F178, T179, R180,G183, E185, D186, K188, P189, K201, V202, D203, E213, E224, T225, G226,K228, E239, E240, T241, H243, K247, N249, I251, R252, 1253, P255, H257,N258, N260, A261, A262, 1263, N264, K265, A266, D276, E277, P278, V280,N282, S283, Y284, D292, K293, E294, N297, I298, K301, F302, G303, S304,Y306, R312, F314, H315, K316, G317, R318, S319, L321, V322, Y325, R327,P329, L330, D332, R333, A334, T335, L337, R338, K341, F342, T343, Y345,N346, H354, E355, G357, R358, Q362, E372, E374, G375, E388, M391, K392,G393, K394, R403, N406, K409, E410, K411, and K413 of the mature FIXpolypeptide set forth in SEQ ID NO:3.

Exemplary modifications that introduce a glycosylation include thoseselected from among modifications corresponding to amino acidreplacements Y1N, Y1N+S3T, S3N+K5S/T, G4T, G4N+L6S/T, K5N+E7T, L6N+E8T,E7N+F9T, F9N+Q11S/T, V10N+G12S/T, Q11N+N13T, G12N+L14S/T, L14N+R16T,E15T, E15N+E17T; R16N+C18S/T, M19N+E21T; E20N+K22T, K22N, S24N+E26T;F25N+E27T; E26N+A28T; E27N+R29T; A28N+E30T; R29N+V31S/T, E30N+F32T;V31N+E33T; F32N+N34T, E33N, T35N+R37S/T, E36T; E36N; R37N, T39N+F41S/T,E40N+W42T, F41N+K43S/T, W42N+Q44S/T, K43N+Y45T; Q44N+V46S/T, Y45N+D47T,V46N+G48S/T, D47N+D49S/T, G48N+Q50S/T, D49N+C51S/T, Q50N+E52S/T,E52N+N54T, S53N+P55S/T, C56S/T, L57N+G59S/T, G59N+561T; G60S/T,S61N+K63S/T, K63N+D65S/T, D65N+N67S/T, I66N+S68S/T, Y69S/T, Y69N+C71S/T,S68N+E70S/T, E70N+W72S/T, W72N+P74S/T, P74N+G76S/T, F75N, G76N+E78T,E78N+K80T, F77T, F77N+G79S/T, G79N+N81S/T, K80N+C82S/T, E83S/T,E83N+D85S/T, L84N+V86S/T, D85N, V86A, V86N+C88S/T, T87N+N89S/T,190N+N92S/T, K91S/T, 190N+N92S/T, K91N+G93S/T, R94S/T, R94N+E96S/T,K100N, A103S/T, S102N+D104S/T, A103N+N105S/T, D104N+K106S/T, V107S/T,K106N+V108S/T, V108N+V110S/T, S111N, E113N+Y115S/T, G114N+R116S/T,R116N+A118S/T, E119N+Q121S/T, K122S/T, Q121N+S123S/T, K122N+C124S/TS123N+E125S/T, E125N+A125S/T, P126N+V128S/T, A127N+P129T, V128N+F130S/T,P129N+P131S/T, F130N+C132S/T, R134N, V135N+V137S/T, S136N, S138N,V137N+Q139T; Q139N, T140N+L142S/T, S141N+L143S/T, K142N, A146N+A148S/T,E147N+V149S/T, T148N+F150S/T, V149N+P151S/T, F150N+D152S/T,P151N+V153S/T, D152N+D154S/T, V153N+Y155S/T, D154N+V156S/T,Y155N+N157S/T, V156N, S158N+E160S/T, T159N+A161S/T, E160N+E162S/T,A161N, E162N+11645/T, T163N+L165S/T, I164N+D166S/T, L165N+N167S/T,D166N+11685/T, 1168N+Q170S/T, T169N, Q170N, S171N+Q173S/T, T172N,Q173N+F175S/T, S174N+N176S/T, F175N+D177S/T, F178S/T, D177N, D177E,F178N+R180S/T, T179N+V181S/T, R180N+V182S/T, G183+E185S/T, G184N+D186T,E185N+A187S/T, D186N+K188S/T, A187N+P189T, K188N+G190S/T, P189N+Q181S/T,G200N+V202T, K201N+D203S/T, K201T, V202N+A204S/T, D203N+F205S/T,E213N+W215S/T, K214T, V223T, E224N+G226S/T, T225N+V227S/T,G226N+K228S/T, V227N+1229T, K228N, H236N+1238T; 1238N+E240T; E239N,E240N+E242S/T, E242N, T241N+H243S/T, H243N+E245S/T, K247N+N249S/T,V250N+R252T, I251S/T, I251N+1253S/T, R252N+I254S/T, I253N+P255S/T,P255N+H257S/T, H257N+Y259S/T, N260S/T, A262S/T, A261N+1263 SIT,A262N+N264S/T, I263N+K265S/T, K265N+N267S/T, A266N+H268S/T,D276N+P278S/T, P278N+V280S/T, E277N+L279S/T, V280N+N282S/T, Y284S/T,S283N+V285S/T, Y284N, D292N+K294S/T, K293N+Y295S/T, E294N, F299S/T,I298N+L300S/T, K301N+G303S/T, F302N, G303N+G305S/T, S304N+Y306S/T,Y306N+S308S/T, R312N+F314S/T, V313N+H315T, F314N+K316S/T, H315N+G317S/T,K316N+R138S/T, G317N, R318N+A320S/T, S319N+L321S/T, A320N+V322T,L321N+L323S/T, V322N+Q324S/T, Y325N+R327S/T, R327N+P329S/T,P329N+V331S/T, L330N+D332S/T, D332N+A334S/T, R333N, A334N+C336S/T,T335N+L337S/T, L337N, R338N, S339N+K341T, T340N+F342T; K341N,F342N+13445/T, T343N+Y345S/T, Y345N+N347S/T, M348S/T, G352N+H354T,F353N, F353N+E355T, H354N+G356S/T, H354V, H3541, E355T, E355N+G357S/T,G356N+R358T, G357N+D359S/T, R358N, Q362N+D364S/T, V370N; T371V; T3711;E372T, E372N+E374S/T, E374N, G375N, W385N+E387T; G386N+E388T,E388N+A390S/T, A390N+K392T, M391N+G393S/T, K392N+K394S/T, K392V, G393T,G393N+Y395S/T, K394N+G396S/T, R403N+V405S/T, 14085/T, K409N+K411S/T,E410N, K411N+K413S/T, and K413N. In some examples, 1, 2, 3, 4, 5, 6, 7,8 or more glycosylation sites are introduced.

Also provided herein are modified FIX polypeptides containing one ormore modifications that eliminate one or more N-glycosylation sitescompared to the unmodified FIX polypeptide. For example, N-glycosylationsites at an amino acid positions corresponding to N157 or N167 of themature FIX polypeptide set forth in SEQ ID NO:3 can be eliminated.Exemplary modifications that eliminate an N-glycosylation site includethose selected from among modifications corresponding to amino acidreplacements N157D, N157Q, N167D and N167Q. In further examples, the FIXpolypeptide contains one or more modifications that eliminate one ormore O-glycosylation sites compared to the unmodified FIX polypeptide.For example, O-glycosylation sites that can be eliminated include thoseamino acid positions corresponding to positions selected from among S53,S61, T159 and T169 of the mature FIX polypeptide set forth in SEQ IDNO:3. Exemplary modifications that eliminate an N-glycosylation siteinclude those selected from among modifications corresponding to aminoacid replacements S53A, S61A, T159A and T169A.

Also provided are modified FIX polypeptides containing one or moremodifications that introduces and/or eliminates one or more sulfationsites compared to the unmodified FIX polypeptide. In one example, themodified FIX polypeptides contain a modification that eliminates asulfation site at an amino acid position corresponding to position Y155of the mature FIX polypeptide set forth in SEQ ID NO:3. Exemplary ofsuch modifications are those that correspond to amino acid replacementsY155H, Y155F and Y155Q.

Provided are modified FIX polypeptides containing one or moremodifications that introduces and/or eliminates one or morephosphorylation sites compared to the unmodified FIX polypeptide. In oneexample, the modified FIX polypeptide contains a modification thateliminates a phosphorylation site at an amino acid positioncorresponding to position S158 of the mature FIX polypeptide set forthin SEQ ID NO:3. Exemplary of such modifications are those thatcorrespond to amino acid replacements S158A, S158D and S158E. Alsoprovided are FIX polypeptides containing one or more modifications thatintroduces and/or eliminates one or more β-hydroxylation sites comparedto the unmodified FIX polypeptide. In one instance, the modified FIXpolypeptides contain a modification that eliminates a β-hydroxylationsite at an amino acid position corresponding to position D64 of themature FIX polypeptide set forth in SEQ ID NO:3. Exemplary of suchmodifications are those that correspond to amino acid replacements D64Nand D64A.

Any of the modified FIX polypeptides provided herein can contain anyother mutations known in the art, such as, for example, one or moremodifications selected from among amino acid replacements Y1A, Y1C, Y1D,Y1E, Y1G, Y1H, Y1K, Y1N, Y1P, Y1Q, Y1R, Y1S, Y1T, S3T, K5A, K51, K5L,K5F, K5E, L6A, L6C, L6D, L6E, L6G, L6H, L6K, L6N, L6P, L6Q, L6R, L6S,L6T, L6M, F9A, F9C, F9D, F9E, F9G, F9H, F9K, F9N, F9P, F9Q, F9R, F9S,F9T, F91, F9M, F9W, V10A, V10C, V10D, V10E, V10G, V10H, V10K, V10N,V10P, V10Q, V10R, V10S, V10T, V10F, V10I, V10K, V10M, V10W, V10Y, Q11E,Q11D, Q11A, Q11C, Q11G, Q11P, G12D, G12E, G12G, G12H, G12K, G12N, G12P,G12Q, G12R, G12S, G12T, N13A, N13C, N13G, N13H, N13P, N13T, L14A, L14C,L14D, L14E, L14G, L14H, L14K, L14N, L14P, L14Q, L14R, L14S, L14T, L14F,L141, L14M, L14V, L14W, L14Y, E15D, E15H, E15P, R16E, R16A, R16C, R16G,R16P, R16T, E17A, E17C, E17G, E17P, E17T, C18D, C18E, C18G, C18H, C18K,C18N, C18P, C18Q, C18R, C18S, C18T, M19A, M19C, M19D, M19E, M19G, M19H,M19K, M19N, M19P, M19Q, M19R, M19S, M19T, M19F, M191, M19M, M19V, M19W,M19Y, E20A, E20C, E20G, E20P, E20T, E21A, E21C, E21G, E21P, K22H, K22P,K22T, S24H, S24P, F25A, F25C, F25D, F25E, F25G, F25H, F25K, F25N, F25P,F25Q, F25R, F25S, F25T, F251, F25M, F25W, F25Y, E26A, E26C, E26G, E26P,E27A, E27C, E27G, E27H, E27P, E27S, E27T, A28C, A28D, A28E, A28G, A28H,A28K, A28N, A28P, A28Q, A28R, A28S, A28T, R29A, R29C, R29G, R29P, R29F,E30D, E30H, E30P, V31A, V31C, V31D, V31E, V31G, V31H, V31K, V31N, V31P,V31Q, V31R, V31S, V31T, V31F, V311, V31W, V31Y, F32A, F32C, F32D, F32E,F32G, F32H, F32K, F32N, F32P, F32Q, F32R, F32S, F32T, E33H, E33N, E33P,E33Q, E33S, E33T, N34E, N34D, N34F, N341, N34L, T35D, T35E, T35A, T35C,T35G, T35P, F41A, F41C, F41D, F41E, F41G, F41H, F41K, F41N, F41P, F41Q,F41R, F41S, F41T, F41M, F41W, F41Y, W42A, W42C, W42D, W42E, W42G, W42H,W42K, W42N, W42P, W42Q, W42R, W42S, W42T, K43A, K43C, K43G, K43P, Q44P,Q44T, Q44, Y45A, Y45C, Y45D, Y45E, Y45G, Y45H, Y45K, Y45N, Y45P, Y45Q,Y45R, Y45S, Y45T, V46A, V46C, V46D, V46E, V46G, V46H, V46K, V46N, V46P,V46Q, V46R, V46S, V46T, V46F, V461, V46M, V46W, V46Y, D47A, D47C, D47G,D47H, D47P, D47T, G48D, G48E, G48P, G48T, D49H, D49P, D49Q, D49T, Q50A,Q50C, Q50D, Q50G, Q50H, Q50P, Q50T, C51D, C51E, C51G, C51H, C51K, C51N,C51P, C51Q, C51R, C51S, C51T, E52P, E52T, S53A, S53C, S53G, S53H, S53P,S53T, N54H, N54P, N54T, L57A, L57C, L57D, L57E, L57G, L57H, L57K, L57N,L57P, L57Q, L57R, L57S, L57T, L57F, L571, L57M, L57W, L57Y, G60C, G60D,G60H, G60P, G60T, C62D, C62H, C62P, K63T, D65H, D65T, 166A, 166C, 166D,166E, 166G, 166H, 166K, 166N, 166P, 166Q, 166R, 166S, 166T, 166M, 166W,166Y, Y69A, Y69C, Y69D, Y69E, Y69G, Y69H, Y69K, Y69N, Y69P, Y69Q, Y69R,Y69S, Y69T, C71H, C71P, W72A, W72C, W72D, W72E, W72G, W72H, W72K, W72N,W72P, W72Q, W72R, W72S, W72T, W721, W72Y, F75A, F75C, F75D, F75E, F75G,F75H, F75K, F75N, F75P, F75Q, F75R, F75S, F75T, F77A, F77C, F77D, F77E,F77G, F77H, F77K, F77N, F77P, F77Q, F77R, F77S, F77T, L84A, L84C, L84D,L84E, L84G, L84H, L84K, L84N, L84P, L84Q, L84R, L84S, L84T, L84M, L84W,L84Y, V86I, V86L, V86M, V86F, V86W, V86Y, V86A, V86C, V86D, V86E, V86G,V86H, V86K, V86N, V86P, V86Q, V86R, V86S, V86T, 190A, 190C, 190D, 190E,190G, 190H, 190K, 190N, 190P, 190Q, 190R, 190S, 190T, 190M, 190W, K91A,K91C, K91G, K91P, N92A, N92C, N92G, N92P, N92T, G93D, G93E, G93H, G93K,G93N, G93P, G93Q, G93R, G93S, G93T, R94A, R94C, R94G, R94P, C95D, C95E,C95G, C95H, C95K, C95N, C95P, C95Q, C95R, C95S, C95T, E96P, E96T, Q97A,Q97C, Q97G, Q97P, F98A, F98C, F98D, F98E, F98G, F98H, F98K, F98N, F98P,F98Q, F98R, F98S, F98T, F98M, F98W, F98Y, K100A, K100C, K100G, K100P,N101H, N101T, A103D, A103E, A103H, A103K, A103N, A103P, A103Q, A103R,A103S, A103T, D104T, K106H, K106P, K106T, V107A, V107C, V107D, V107E,V107G, V107H, V107K, V107N, V107P, V107Q, V107R, V107S, V107T, V108A,V108C, V108D, V108E, V108G, V108H, V108K, V108N, V108P, V108Q, V108R,V108S, V108T, V108F, V108M, V108W, V108Y, S110A, S110C, S110G, S110P,C111D, C111E, C111H, C111K, C111N, C111P, C111Q, C111R, C111S, C111T,T112A, T112C, T112G, T112P, E113D, E113H, E113P, G114D, G114E, G114H,G114K, G114N, G114P, G114Q, G114R, G114S, G114T, Y115A, Y115C, Y115D,Y115E, Y115G, Y115H, Y115K, Y115N, Y115P, Y115Q, Y115R, Y115S, Y115T,Y115M, Y115W, R116P, R116T, L117A, L117C, L117D, L117E, L117G, L117H,L117K, L117N, L117P, L117Q, L117R, L117S, L117T, A118D, A118E, A118H,A118K, A118N, A118P, A118Q, A118R, A118S, A118T, N120D, N120H, N120P,Q121T, S123H, S123T, V128A, V128C, V128D, V128E, V128G, V128H, V128K,V128N, V128P, V128Q, V128R, V128S, V128T, F130A, F130C, F130D, F130E,F130G, F130H, F130K, F130N, F130P, F130Q, F130R, F130S, F130T, V135A,V135C, V135D, V135E, V135G, V135H, V135K, V135N, V135P, V135Q, V135R,V135S, V135T, V135W, V135Y, V137A, V137C, V137D, V137E, V137G, V137H,V137K, V137N, V137P, V137Q, V137R, V137S, V137T, V137M, V137W, V137Y,S138H, S138T, T140D, T140H, S141T, K142H, K142P, L143A, L143C, L143D,L143E, L143G, L143H, L143K, L143N, L143P, L143Q, L143R, L143S, L143T,L143F, L1431, L143M, L143V, L143W, L143Y, R145H, R145P, R145T, A146P,A146T, T148H, T148P, V149A, V149C, V149D, V149E, V149G, V149H, V149K,V149N, V149P, V149Q, V149R, V149S, V149T, V149F, V149I, V149M, V149W,V149Y, F150A, F150C, F150D, F150E, F150G, F150H, F150K, F150N, F150P,F150Q, F150R, F150S, F150T, F150M, F150W, F150Y, D152A, D152C, D152G,D152P, D152S, D152T, V153A, V153C, V153D, V153E, V153G, V153H, V153K,V153N, V153P, V153Q, V153R, V153S, V153T, V153F, V1531, V153M, V153W,V153Y, D154A, D154C, D154G, D154P, D154Q, D154S, Y155A, Y155C, Y155D,Y155E, Y155G, Y155H, Y155K, Y155N, Y155P, Y155Q, Y155R, Y155S, Y155T,Y155M, Y155V, Y155W, V156A, V156C, V156D, V156E, V156G, V156H, V156K,V156N, V156P, V156Q, V156R, V156S, V156T, V1561, V156M, V156W, V156Y,N157A, N157C, N157G, N157H, N157P, N157Q, N157T, S158H, S158P, S158T,T159A, T159C, T159G, T159P, E160A, E160C, E160G, E160P, A161C, A161D,A161E, A161H, A161K, A161N, A161P, A161Q, A161R, A161S, A161T, E162P,E162T, T163A, T163C, T163G, T163P, I164A, I164C, I164D, 1164E, I164G,I164H, I164K, I164N, I164P, I164Q, I164R, I164S, I164T, L165A, L165C,L165D, L165E, L165G, L165H, L165K, L165N, L165P, L165Q, L165R, L165S,L165T, L165M, L165W, L165Y, I168A, I168C, I168D, 1168E, I168G, I168H,I168K, I168N, I168P, I168Q, I168R, I168S, I168T, F175A, F175C, F175D,F175E, F175G, F175H, F175K, F175N, F175P, F175Q, F175R, F175S, F175T,F178A, F178C, F178D, F178E, F178G, F178H, F178K, F178N, F178P, F178Q,F178R, F178S, F178T, F178M, F178W, F178Y, T179A, T179C, T179G, T179P,R180A, R180C, R180D, R180G, R180H, R180P, V181A, V181C, V181D, V181E,V181G, V181H, V181K, V181N, V181P, V181Q, V181R, V181S, V181T, V181F,V181I, V181M, V181W, V181Y, V182A, V182C, V182D, V182E, V182G, V182H,V182K, V182N, V182P, V182Q, V182R, V182S, V182T, V182F, V182I, V182M,V182W, V182Y, G183D, G183E, G183H, G183K, G183N, G183P, G183Q, G183S,G183T, G184D, G184E, G184H, G184K, G184N, G184P, G184Q, G184R, G184S,G184T, E185A, E185C, E185G, E185H, E185P, E185T, D186A, D186C, D186G,D186H, D186P, D186T, A187C, A187D, A187E, A187G, A187H, A187K, A187N,A187P, A187Q, A187R, A187S, A187T, K188A, K188C, K188G, K188H, K188P,K188T, G190D, G190E, G190H, G190K, G190N, G190P, G190Q, G190R, G190S,G190T, F192A, F192C, F192D, F192E, F192G, F192H, F192K, F192N, F192P,F192Q, F192R, F192S, F192T, F192W, F192Y, W194A, W194C, W194D, W194E,W194G, W194H, W194K, W194N, W194P, W194Q, W194R, W194S, W194T, Q195H,Q195P, Q195T, V196A, V196C, V196D, V196E, V196G, V196H, V196K, V196N,V196P, V196Q, V196R, V196S, V196T, V196F, V1961, V196M, V196W, V196Y,V197A, V197C, V197D, V197E, V197G, V197H, V197K, V197N, V197P, V197Q,V197R, V197S, V197T, V197F, V1971, V197M, V197W, V197Y, L198A, L198C,L198D, L198E, L198G, L198H, L198K, L198N, L198P, L198Q, L198R, L198S,L198T, L1981, L198Y, N199A, N199C, N199G, N199H, N199P, N199S, N199T,G200P, G200T, K201A, K201C, K201D, K201E, K201G, K201H, K201N, K201P,K201Q, K201S, K201T, V202A, V202C, V202D, V202E, V202G, V202H, V202K,V202N, V202P, V202Q, V202R, V202S, V202T, V202F, V2021, V202M, V202W,V202Y, D203A, D203C, D203G, D203P, D203T, A204C, A204D, A204E, A204G,A204H, A204K, A204N, A204P, A204Q, A204R, A204S, A204T, F205A, F205C,F205D, F205E, F205G, F205H, F205K, F205N, F205P, F205Q, F205R, F205S,F205T, F205M, F205V, F205W, F205Y, G207H, G207P, G208C, G208D, G208E,G208H, G208K, G208N, G208P, G208Q, G208R, G208S, G208T, S209A, S209C,S209G, S209P, 1210A, 1210C, 1210D, I210E, I210G, I210H, I210K, I210N,I210P, I210Q, I210R, I210S, I210T, I210F, I210W, I210Y, V211A, V211C,V211D, V211E, V211G, V211H, V211K, V211N, V211P, V211Q, V211R, V211S,V211T, V211F, V211I, V211M, V211W, N212A, N212C, N212G, N212P, E213H,E213P, E213S, E213T, K214T, W215A, W215C, W215D, W215E, W215G, W215H,W215K, W215N, W215P, W215Q, W215R, W215S, W215T, I216A, I216C, I216D,I216E, I216G, I216H, I216K, I216N, I216P, I216Q, I216R, I216S, I216T,V217A, V217C, V217D, V217E, V217G, V217H, V217K, V217N, V217P, V217Q,V217R, V217S, V217T, V217I, V217Y, A219H, A219P, A219T, V223A, V223C,V223D, V223E, V223G, V223H, V223K, V223N, V223P, V223Q, V223R, V223S,V223T, V223M, V223W, V223Y, G226P, V227A, V227C, V227D, V227E, V227G,V227H, V227K, V227N, V227P, V227Q, V227R, V227S, V227T, V227F, V227I,V227M, V227W, V227Y, K228A, K228C, K228G, K228H, K228P, I229A, I229C,I229D, I229E, I229G, I229H, I229K, I229N, I229P, I229Q, I229R, I229S,I229T, I229M, I229W, I229Y, T230A, T230C, T230G, T230P, V231A, V231C,V231D, V231E, V231G, V231H, V231K, V231N, V231P, V231Q, V231R, V231S,V231T, V232A, V232C, V232D, V232E, V232G, V232H, V232K, V232N, V232P,V232Q, V232R, V232S, V232T, V232F, V2321, V232M, V232W, V232Y, A233C,A233D, A233E, A233G, A233H, A233K, A233N, A233P, A233Q, A233R, A233S,A233T, A233V, G234D, G234E, G234H, G234K, G234N, G234P, G234Q, G234R,G234S, G234T, E235H, E235N, E235P, E235Q, E235S, E235T, H236A, H236C,H236G, H236P, N237A, N237C, N237G, N237P, N237T, I238A, I238C, I238D,I238E, I238G, I238H, I238K, I238N, I238P, I238Q, I238R, I238S, I238T,E239A, E239C, E239G, E239P, E240H, E240T, V250A, V250C, V250D, V250E,V250G, V250H, V250K, V250N, V250P, V250Q, V250R, V250S, V250T, V250M,V250W, V250Y, I251A, I251C, I251D, 1251E, I251G, I251H, I251K, I251N,I251P, I251Q, I251R, I251S, I251T, I253A, I253C, I253D, I253E, I253G,I253H, I253K, I253N, I253P, I253Q, I253R, I253S, I253T, I253M, I253W,I253Y, I254A, I254C, I254D, I254E, I254G, I254H, I254K, I254N, I254P,I254Q, I254R, I254S, I254T, P255H, H256P, H256T, H257A, H257C, H257G,H257P, N258P, N258T, Y259A, Y259C, Y259D, Y259E, Y259G, Y259H, Y259K,Y259N, Y259P, Y259Q, Y259R, Y259S, Y259T, Y259M, Y259W, Y259F, N260A,N260C, N260G, N260P, A261D, A261E, A261H, A261K, A261N, A261P, A261Q,A261R, A261S, A261T, A262C, A262D, A262E, A262G, A262H, A262K, A262N,A262P, A262Q, A262R, A262S, A262T, I263A, I263C, I263D, I263E, I263G,I263H, I263K, I263N, I263P, I263Q, I263R, I263S, I263T, I263M, I263V,I263W, I263Y, N264A, N264C, N264D, N264G, N264H, N264P, K265A, K265C,K265G, K265H, K265P, K265T, Y266A, Y266C, Y266D, Y266E, Y266G, Y266H,Y266K, Y266N, Y266P, Y266Q, Y266R, Y266S, Y266T, Y266M, Y266W, N267A,N267C, N267G, N267H, N267P, N267T, H268P, D269A, D269C, D269E, D269G,D269H, D269N, D269P, D269Q, D269S, D269T, I270A, I270C, I270D, I270E,I270G, I270H, I270K, I270N, I270P, I270Q, I270R, I270S, I270T, I270M,I270W, A271C, A271D, A271E, A271G, A271H, A271K, A271N, A271P, A271Q,A271R, A271S, A271T, L272A, L272C, L272D, L272E, L272G, L272H, L272K,L272N, L272P, L272Q, L272R, L272S, L272T, L272F, L273A, L273C, L273D,L273E, L273G, L273H, L273K, L273N, L273P, L273Q, L273R, L273S, L273T,L273F, L273I, L273M, L273V, L273W, L273Y, E274A, E274C, E274G, E274P,E274T, L275A, L275C, L275D, L275E, L275G, L275H, L275K, L275N, L275P,L275Q, L275R, L275S, L275T, L275W, L275Y, D276P, D276S, D276T, E277A,E277C, E277G, E277P, E277V, E277N, E277D, E277E, E277Q, E277H, E2771,E277L, E277M, E277F, E277S, E277T, E277W, E277Y, P278T, L279A, L279C,L279D, L279E, L279G, L279H, L279K, L279N, L279P, L279Q, L279R, L279S,L279T, L279I, L279Y, V280A, V280C, V280D, V280E, V280G, V280H, V280K,V280N, V280P, V280Q, V280R, V280S, V280T, V280F, V2801, V280W, V280Y,L281A, L281C, L281D, L281E, L281G, L281H, L281K, L281N, L281P, L281Q,L281R, L281S, L281T, L281F, L281I, L281V, L281W, L281Y, S283A, S283C,S283G, S283P, Y284A, Y284C, Y284D, Y284E, Y284G, Y284H, Y284K, Y284N,Y284P, Y284Q, Y284R, Y284S, Y284T, Y284M, V285A, V285C, V285D, V285E,V285G, V285H, V285K, V285N, V285P, V285Q, V285R, V285S, V285T, V285M,V285W, V285Y, T286A, T286C, T286G, T286P, I288A, I288C, I288D, I288E,I288G, I288H, I288K, I288N, I288P, I288Q, I288R, I288S, I288T, C289D,C289H, C289P, I290A, I290C, I290D, I290E, I290G, I290H, I290K, I290N,I290P, I290Q, I290R, I290S, I290T, I290Y, A291D, A291E, A291H, A291K,A291N, A291P, A291Q, A291R, A291S, A291T, D292A, D292C, D292G, D292P,D292T, K293H, K293P, K293T, Y295A, Y295C, Y295D, Y295E, Y295G, Y295H,Y295K, Y295N, Y295P, Y295Q, Y295R, Y295S, Y295T, Y295W, T296A, T296C,T296G, T296P, N297A, N297C, N297G, N297P, I298A, I298C, I298D, I298E,I298G, I298H, I298K, I298N, I298P, I298Q, I298R, I298S, I298T, F299A,F299C, F299D, F299E, F299G, F299H, F299K, F299N, F299P, F299Q, F299R,F299S, F299T, L300A, L300C, L300D, L300E, L300G, L300H, L300K, L300N,L300P, L300Q, L300R, L300S, L300T, L300F, L300I, L300M, L300V, L300W,L300Y, K301A, K301C, K301G, K301P, K301T, F302A, F302C, F302D, F302E,F302G, F302H, F302K, F302N, F302P, F302Q, F302R, F302S, F302T, G303H,G303P, G303T, S304A, S304C, S304G, S304P, S304T, G305D, G305E, G305H,G305N, G305P, G305Q, G305S, G305T, Y306A, Y306C, Y306D, Y306E, Y306G,Y306H, Y306K, Y306N, Y306P, Y306Q, Y306R, Y306S, Y306T, V307A, V307C,V307D, V307E, V307G, V307H, V307K, V307N, V307P, V307Q, V307R, V307S,V307T, S308P, S308T, W310A, W310C, W310D, W310E, W310G, W310H, W310K,W310N, W310P, W310Q, W310R, W310S, W310T, G311H, V313A, V313C, V313D,V313E, V313G, V313H, V313K, V313N, V313P, V313Q, V313R, V313S, V313T,F314A, F314C, F314D, F314E, F314G, F314H, F314K, F314N, F314P, F314Q,F314R, F314S, F314T, F314M, F314W, F314Y, H315A, H315C, H315G, H315P,K316A, K316C, K316G, K316P, G317C, G317D, G317E, G317H, G317K, G317N,G317P, G317Q, G317R, G317S, G317T, R318A, R318C, R318G, R318P, S319D,S319H, S319N, S319P, S319Q, A320C, A320D, A320E, A320G, A320H, A320K,A320N, A320P, A320Q, A320R, A320S, A320T, L321A, L321C, L321D, L321E,L321G, L321H, L321K, L321N, L321P, L321Q, L321R, L321S, L321T, V322A,V322C, V322D, V322E, V322G, V322H, V322K, V322N, V322P, V322Q, V322R,V322S, V322T, V322W, V322Y, L323A, L323C, L323D, L323E, L323G, L323H,L323K, L323N, L323P, L323Q, L323R, L323S, L323T, L323F, L323I, L323M,L323V, L323W, L323Y, Q324A, Q324C, Q324G, Q324P, Y325A, Y325C, Y325D,Y325E, Y325G, Y325H, Y325K, Y325N, Y325P, Y325Q, Y325R, Y325S, Y325T,Y325W, L326A, L326C, L326D, L326E, L326G, L326H, L326K, L326N, L326P,L326Q, L326R, L326S, L326T, L326F, L326I, L326M, L326V, L326W, L326Y,R327A, R327C, R327G, R327H, R327P, V328A, V328C, V328D, V328E, V328G,V328H, V328K, V328N, V328P, V328Q, V328R, V328S, V328T, V328F, V3281,V328M, V328W, V328Y, L330A, L330C, L330D, L330E, L330G, L330H, L330K,L330N, L330P, L330Q, L330R, L330S, L330T, L330F, L330I, L330V, L330W,L330Y, V331A, V331C, V331D, V331E, V331G, V331H, V331K, V331N, V331P,V331Q, V331R, V331S, V331T, V331F, V331I, V331M, V331W, V331Y, D332A,D332C, D332G, D332P, R333A, R333C, R333D, R333E, R333G, R333H, R333N,R333P, R333Q, R333R, R333S, R333T, A334C, A334D, A334E, A334G, A334H,A334K, A334N, A334P, A334Q, A334R, A334S, A334T, T335A, T335C, T335G,T335P, C336D, C336E, C336H, C336K, C336N, C336P, C336Q, C336R, C336S,C336T, L337A, L337C, L337D, L337E, L337G, L337H, L337K, L337N, L337P,L337Q, L337R, L337S, L337T, R338A, R338E, R338V, R338T, R338C, R338G,R338P, R3381, R338F, R338W, R338S, S339P, S339T, K341A, K341C, K341G,K341P, F342A, F342C, F342D, F342E, F342G, F342H, F342K, F342N, F342P,F342Q, F342R, F342S, F342T, F342M, F342W, T343A, T343C, T343G, T343P,I344A, I344C, I344D, I344E, I344G, I344H, I344K, I344N, I344P, I344Q,I344R, I344S, I344T, Y345F, Y345A, Y345C, Y345D, Y345E, Y345G, Y345H,Y345K, Y345N, Y345P, Y345Q, Y345R, Y345S, Y345T, Y345M, Y345W, N346A,N346C, N346G, N346P, N347H, N347P, M348A, M348C, M348D, M348E, M348G,M348H, M348K, M348N, M348P, M348Q, M348R, M348S, M348T, F349A, F349C,F349D, F349E, F349G, F349H, F349K, F349N, F349P, F349Q, F349R, F349S,F349T, F349I, F349M, F349W, F349Y, C350D, C350H, C350P, C350T, A351E,A351H, A351N, A351P, A351Q, A351R, A351S, A351T, G352A, G352C, G352P,F353A, F353C, F353D, F353E, F353G, F353H, F353K, F353N, F353P, F353Q,F353R, F353S, F353T, F353I, F353M, F353W, H354A, H354C, H354G, H354P,E355A, E355C, E355D, E355G, E355H, E355K, E355N, E355P, E355Q, E355S,E355T, G356D, G356E, G356H, G356K, G356N, G356P, G356Q, G356R, G356S,G356T, G357D, G357E, G357H, G357K, G357N, G357P, G357Q, G357R, G357S,G357T, R358D, R358E, R358H, R358K, R358N, R358P, R358Q, R358R, R358S,R358T, D359A, D359C, D359G, D359P, D359Q, D359S, D359T, S360A, S360C,S360G, S360P, C361D, C361E, C361H, C361K, C361N, C361P, C361Q, C361R,C361S, C361T, V370A, V370C, V370D, V370E, V370G, V370H, V370K, V370N,V370P, V370Q, V370R, V370S, V370T, V370W, V370Y, V373A, V373C, V373D,V373E, V373G, V373H, V373K, V373N, V373P, V373Q, V373R, V373S, V373T,V373F, V3731, V373M, V373W, E374A, E374C, E374G, E374P, G375H, S377A,S377C, S377G, S377P, F378A, F378C, F378D, F378E, F378G, F378H, F378K,F378N, F378P, F378Q, F378R, F378S, F378T, F378W, L379A, L379C, L379D,L379E, L379G, L379H, L379K, L379N, L379P, L379Q, L379R, L379S, L379T,L379I, L379M, L379W, L379Y, T380A, T380C, T380G, T380P, G381D, G381E,G381H, G381K, G381N, G381P, G381Q, G381R, G381S, G381T, I382A, I382C,I382D, I382E, I382G, I382H, I382K, I382N, I382P, I382Q, I382R, I382S,I382T, I382M, I382W, I382Y, I383A, I383C, I383D, I383E, I383G, I383H,I383K, I383N, I383P, I383Q, I383R, I383S, I383T, I383V, S384A, S384C,S384G, S384P, W385A, W385C, W385D, W385E, W385G, W385H, W385K, W385N,W385P, W385Q, W385R, W385S, W385T, W385M, E387A, E387C, E387G, E387H,E387P, E387T, E388H, E388N, E388G, E388P, E388Q, E388T, A390C, A390D,A390E, A390G, A390H, A390K, A390N, A390P, A390Q, A390R, A390S, M391A,M391C, M391D, M391E, M391G, M391H, M391K, M391N, M391P, M391Q, M391R,M391S, M391T, M391F, M391I, M391W, M391Y, K392A, K392C, K392G, K392P,G393C, G393D, G393E, G393H, G393K, G393N, G393P, G393Q, G393R, G393S,G393T, Y395A, Y395C, Y395D, Y395E, Y395G, Y395H, Y395K, Y395N, Y395P,Y395Q, Y395R, Y395S, Y395T, Y398A, Y398C, Y398D, Y398E, Y398G, Y398H,Y398K, Y398N, Y398P, Y398Q, Y398R, Y398S, Y398T, K400H, V401A, V401C,V401D, V401E, V401G, V401H, V401K, V401N, V401P, V401Q, V401R, V401S,V401T, V401F, V4011, V401M, V401W, V401Y, S402A, S402C, S402G, S402P,R403A, R403C, R403G, R403P, R403T, Y404A, Y404C, Y404D, Y404E, Y404G,Y404H, Y404K, Y404N, Y404P, Y404Q, Y404R, Y404S, Y404T, V405A, V405C,V405D, V405E, V405G, V405H, V405K, V405N, V405P, V405Q, V405R, V405S,V405T, V405W, V405Y, N406F, N406H, N4061, N406L, N406P, N406W, N406Y,W407D, W407E, W407F, W407H, W4071, W407K, W407N, W407P, W407Q, W407R,W407S, W407T, W407Y, I408D, I408E, I408H, I408K, I408N, I408P, I408Q,I408R, I408S, I408T, K409F, K409H, K4091, K409P, K409T, K409V, K409W,K409Y, E410H, K411A, K411C, K411G, K4111, K411P, K411T, K411V, K411W,K411Y, K413T, Y1I, S3Q, S3H, S3N, G4Q, G4H, G4N, K5N, K5Q, L61, L6V,E7Q, E7H, E7N, E8Q, E8H, E8N, F9V, E15Q, E15N, R16H, R16Q, E17Q, E17H,E17N, E20Q, E20H, E20N, E21Q, E21H, E21N, K22N, K22Q, S24Q, S24N, F25V,E26Q, E26H, E26N, E27Q, E27N, R29H, R29Q, E30Q, E30N, F321, F32V, T35Q,T35H, T35N, E36Q, E36H, E36N, R37H, R37Q, T38Q, T38H, T38N, T39Q, T39H,T39N, E40Q, E40H, E40N, F411, F41V, K43N, K43Q, Y451, D47N, D47Q, G48Q,G48H, G48N, D49N, E52Q, E52H, E52N, S53Q, S53N, P55A, P55S, L57V, N58Q,N58S, G59Q, G59H, G59N, G60Q, G60N, S61Q, S61H, S61N, K63N, K63Q, D64N,D64Q, D65N, D65Q, S68Q, S68H, S68N, Y691, E70Q, E70H, E70N, P74A, P74S,F751, F75V, G76Q, G76H, G76N, F771, F77V, E78Q, E78H, E78N, G79Q, G79H,G79N, K80N, K80Q, E83Q, E83H, E83N, L841, L84V, D85N, D85Q, T87Q, T87H,T87N, K91N, K91Q, N92Q, N92S, R94H, R94Q, E96Q, E96H, E96N, F981, F98V,K100N, K100Q, S102Q, S102H, S102N, D104N, D104Q, K106N, K106Q, S110Q,S110H, S110N, T112Q, T112H, T112N, E113Q, E113N, Y115I, R116H, R116Q,L1171, L117V, E119Q, E119H, E119N, K122N, K122Q, S123Q, S123N, E125Q,E125H, E125N, P126A, P126S, A127Q, A127H, A127N, P129A, P129S, P131A,P131S, G133Q, G133H, G133N, R134H, R134Q, S136Q, S136H, S136N, S138Q,S138N, T140Q, T140N, S141Q, S141H, S141N, K142N, K142Q, T144Q, T144H,T144N, R145Q, A146Q, A146H, A146N, E147Q, E147H, E147N, T148Q, T148N,P151A, P151S, D152N, D152Q, D154N, Y1551, S158Q, S158N, T159Q, T159H,T159N, E160Q, E160H, E160N, E162Q, E162H, E162N, T163Q, T163H, T163N,L165I, L165V, D166N, D166Q, T169Q, T169H, T169N, S171Q, S171H, S171N,T172Q, T172H, T172N, S174Q, S174H, S174N, F175I, F175V, D177N, D177Q,F1781, F178V, T179Q, T179H, T179N, R180Q, E185Q, E185N, D186N, D186Q,K188N, K188Q, P189A, P189S, F1921, F192V, F192IH, P193A, P193S, W1941,L198V, N199Q, G200Q, G200H, G200N, D203N, D203Q, F205I, G207Q, G207N,S209Q, S209H, S209N, E213Q, E213N, K214N, K214Q, T218Q, T218H, T218N,A219Q, A219N, A220Q, A220H, A220N, E224Q, E224H, E224N, T225Q, T225H,T225N, G226Q, G226H, G226N, K228N, K228Q, T230Q, T230H, T230N, E239Q,E239H, E239N, E240Q, E240N, T241Q, T241H, T241N, E242Q, E242H, E242N,T244Q, T244H, T244N, E245Q, E245H, E245N, K247N, K247Q, R248H, R248Q,R252H, R252Q, P255A, P255S, Y259I, K265N, K265Q, Y266I, L272I, L272V,E274Q, E274H, E274N, L275I, L275V, D276N, D276Q, E277Q, E277H, E277N,P278A, P278S, L279V, S283Q, S283H, S283N, Y284I, T286Q, T286H, T286N,P287A, P287S, D292N, D292Q, K293N, K293Q, E294Q, E294H, E294N, Y295I,T296Q, T296H, T296N, F299I, F299V, K301N, K301Q, F302I, F302V, G303Q,G303N, S304Q, S304H, S304N, Y306I, S308Q, S308H, S308N, G309Q, G309H,G309N, G311Q, G311N, R312H, R312Q, F314I, F314V, K316N, K316Q, R318H,R318Q, L321I, L321V, Y325I, R327Q, P329A, P329S, D332N, D332Q, T335Q,T335H, T335N, L337I, L337V, R338H, R338Q, S339Q, S339H, S339N, T340Q,T340H, T340N, K341N, K341Q, F342I, F342V, T343Q, T343H, T343N, Y345I,M348I, M348V, F349V, G352Q, G352H, G352N, F353V, D359N, S360Q, S360H,S360N, G363Q, G363H, G363N, D364N, D364Q, S365Q, S365H, S365N, G366Q,G366H, G366N, G367Q, G367H, G367N, P368A, P368S, T371Q, T371H, T371N,E372Q, E372H, E372N, E374Q, E374H, E374N, G375Q, G375N, T376Q, T376H,T376N, S377Q, S377H, S377N, F378I, F378V, L379V, T380Q, T380H, T380N,S384Q, S384H, S384N, G386Q, G386H, G386N, E387Q, E387N, M391V, K392N,K392Q, K394N, K394Q, Y395I, G396Q, G396H, G396N, I397Q, I397H, I397N,Y398I, T399Q, T399H, T399N, K400N, K400Q, S402Q, S402H, S402N, R403H,R403Q, Y404I, K409N, K409Q, E410Q, E410N, K411N, K411Q, T412Q, T412H,T412N, K413N, K413Q, L414I, L414V, T415Q, T415H, T415N, R252A, H268A,K293A, K400A, R403A, R403E and K411A.

In some instances, the modified FIX polypeptides provided herein exhibitincreased resistance to antithrombin III, heparin and/or theAT-III/heparin complex compared with the unmodified FIX polypeptide. Forexample, the modified FIX polypeptides can exhibit at least or about 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 200%, 300%,400%, 500% or more increased resistance to antithrombin III and/orheparin compared with the unmodified FIX polypeptide. In furtherinstances, the modified FIX polypeptides exhibit increased catalyticactivity compared with the unmodified FIX polypeptide. This can be inthe presence or absence of FVIIIa. For example, the modified FIXpolypeptides can exhibit at least or about 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, 100%, 200%, 300%, 400%, 500% or more catalyticactivity compared to an unmodified FIX polypeptide.

The modified FIX polypeptides further can exhibit improvedpharmacokinetic properties compared with the unmodified FIX polypeptide,such as, for example, decreased clearance (e.g. at least or about 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the clearance ofan unmodified FIX polypeptide), altered volume of distribution (e.g.decreased by at least or about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% of the volume of distribution of an unmodified FIXpolypeptide, or increased by at least or about 1%, 2%, 3%, 4%, 5%, 6%,7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, 100%, 200%, 300%, 400%, 500% or more ofthe volume of distribution of an unmodified FIX polypeptide), increasedin vivo recovery (e.g. by at least or about 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, 100%, 200%, 300%, 400%, 500% or more of the invivo recovery of an unmodified FIX polypeptide), increased totalmodified FIX polypeptide exposure in vivo (e.g. increased by at least orabout 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 200%,300%, 400%, 500% or more of the total exposure in vivo an unmodified FIXpolypeptide), increased serum half-life (e.g. by at least or about 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 200%, 300%,400%, 500% or more of the serum half-life an unmodified FIXpolypeptide), and/or increased mean resonance time (MRT) compared to theunmodified FIX polypeptide (e.g. increased by at least or about 1%, 2%,3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 200%, 300%, 400%,500% or more of the MRT in vivo an unmodified FIX polypeptide). In someinstances, wherein the improved pharmacokinetic property is increasedserum half-life, the serum half life is α, β or γ phase.

In some instances, the modified FIX polypeptides provided herein exhibitincreased procoagulant activity compared with the unmodified FIXpolypeptide, such as, for example, at least or about 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 200%, 300%, 400%, 500% or morethan the procoagulant activity of an unmodified FIX polypeptide.

In some examples, the unmodified FIX polypeptide has a sequence of aminoacids set forth in SEQ ID NO:3. Thus, provided herein are modified FIXpolypeptides having a sequence of amino acids set forth in any of SEQ IDNOS: 75-272. In other examples, the unmodified FIX polypeptide is avariant of the polypeptide set forth in SEQ ID NO:3, such as an allelicor species variant having 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the polypeptide setforth in SEQ ID NO: 3, excluding the modification(s).

In some instances, the provided modified FIX polypeptides are humanpolypeptides. In other instances, they are non-human polypeptides. Infurther examples, the modified FIX polypeptides are mature polypeptides.Also provided are single chain and two-chain FIX polypeptides, andactive or activated FIX polypeptides. In some examples, activation iseffected by proteolytic cleavage by Factor IX (FIXa) or the TissueFactor/Factor VIIa complex.

In some examples, the provided modified FIX polypeptides have only theprimary sequence modified. In other examples, a chemical modification ora post-translational modification is contained (e.g. the modified FIXpolypeptides are glycosylated, carboxylated, hydroxylated, sulfated,phosphorylated, albuminated, or conjugated to a polyethylene glycol(PEG) moiety). Also provided are chimeric and fusion FIX polypeptides.

The modified FIX polypeptides provided herein can contain 1, 2, 3, 4, 5,6, 7, 8, 9, 10 or more modifications, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,20, 30, 40, 50 or 60 or more modifications, so long as the polypeptideretains at least one FIX activity (e.g. Factor VIIIa binding, Factor Xbinding, phospholipid binding, and/or coagulant activity) of theunmodified FIX polypeptide. For example, the modified FIX polypeptidecan retain at least about or 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, 200%, 300%,400%, 500%, or more of an activity of the unmodified FIX polypeptide. Insome examples, the activities that are retained are increased comparedto the unmodified FIX polypeptide. In other examples, the activitiesthat are retained are decreased compared to the unmodified FIXpolypeptide. The activities can be measured in vitro, ex vivo or invivo.

Provided herein are nucleic acid molecules containing a sequence ofnucleotides encoding any of the provided modified FIX polypeptides. Alsoprovided are vectors containing the nucleic acid molecules. The vectorcan be, for example, a prokaryotic vector, viral vector (e.g. anadenovirus, an adeno-associated-virus, a retrovirus, a herpes virus, alentivirus, a poxvirus, or a cytomegalovirus), or a eukaryotic vector(e.g. a mammalian vector). Also provided are cells containing thesevectors. The cell can be, for example, a eukaryotic cell, such as amammalian cell (e.g. baby hamster kidney cells (BHK-21) or 293 cells orCHO cells). Typically, the cell expresses the modified FIX polypeptide.Thus, also provided are modified FIX polypeptides that are produced byany of the cells provided herein.

Provided are pharmaceutical compositions, containing a therapeuticallyeffective concentration or amount of a modified FIX polypeptide providedherein, in a pharmaceutically acceptable vehicle. In some examples, thepharmaceutical composition is formulated for local, systemic, or topicaladministration, such as oral, nasal, pulmonary, buccal, transdermal,subcutaneous, intraduodenal, enteral, parenteral, intravenous, orintramuscular administration. In further examples, it is formulated forcontrolled-release or for single-dosage administration.

Provided are methods in which a subject is treated by administering theprovided pharmaceutical compositions, wherein the subject has a diseaseor condition that is treated by administration of FIX or a procoagulant.In some instances, the disease or condition is treated by administrationof active FIX (FIXa) or FIX that is not activated. In some examples,treatment with the pharmaceutical composition ameliorates or alleviatesthe symptoms associated with the disease or condition. Also provided aremethods that contain a step of monitoring the subject for changes in thesymptoms associated with disease or condition that is treated byadministration of FIX or a procoagulant.

The disease or condition to be treated using the methods can be selectedfrom among blood coagulation disorders, hematologic disorders,hemorrhagic disorders, hemophilias, and bleeding disorders. In someexamples, the hemophilia is hemophilia B. The methods also can involveadministering one or more additional coagulation factors, such as, forexample, plasma purified or recombinant coagulation factors,procoagulants, such as vitamin K, vitamin K derivative and protein Cinhibitors, plasma, platelets, red blood cells or corticosteroids.

Also provided are articles of manufacture, containing packaging materialand a pharmaceutical composition containing a provided modified FIXpolypeptide contained within the packaging material. The modified FIXpolypeptide is effective for treatment of a disease treatable byadministration of FIX or a procoagulant, and the packaging materialincludes a label that indicates that the modified FIX polypeptide isused for treatment of a disease treatable by administration of FIX or aprocoagulant.

Kits containing any of the pharmaceutical compositions provided herein,a device for administration of the composition and, optionally,instructions for administration also are provided.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the coagulation cascade. The figure shows the intrinsicpathway and the extrinsic pathway of coagulation for the independentproduction of FXa and convergence of the pathways to a common pathway togenerate thrombin and fibrin for the formation of a clot. These pathwaysare interconnected. The figure depicts the order of molecules involvedin the activation cascade in which a zymogen is converted to anactivated protease by cleavage of one or more peptide bonds. Theactivated protease then serves as the activating protease for the nextzymogen molecule in the cascade, ultimately resulting in clot formation.

FIG. 2 depicts the cell based model of coagulation (see e.g. Hoffman etal. (2001) Thromb Haemost 85:958-965). The figure depicts thecoagulation events as being separated into three phases, whereinitiation of coagulation is effected by the activation of FX to FXa bythe TF/FVIIa complex on the TF-bearing cell, resulting in the generationof a small amount of thrombin after activation by FXa/FVa. Amplificationtakes place when thrombin binds to and activates the platelets, andinitiates the activation of sufficient quantities of the appropriatecoagulation factors to form the FVIIIa/FIXa and FVa/FXa complexes.Propagation of coagulation occurs on the surface of large numbers ofactivated platelets at the site of injury, resulting in a burst ofthrombin generation that is sufficiently large to generate enough fibrinfrom fibrinogen to establish a clot at the site of injury.

FIGS. 3A-3D are an alignment of various Factor IX polypeptides,including species variants and modified Factor IX polypeptides (SEQ IDNOS:2-5, 14, 20, 172, 267, 247, 325, 346-347, 360, 365-366, 406). Alsoincluded are SEQ ID NO:6 from U.S. Pat. No. 7,700,734 containingmutations V86A/E277A/R338A and SEQ ID NO:2 from U.S. Pat. No. 7,125,841.A “*” means that the residues or nucleotides in that column areidentical in all sequences in the alignment, a “:” means that conservedsubstitutions have been observed, and a “.” means that semi-conservedsubstitutions are observed. As described herein, residues correspondingto positions in SEQ ID NO:3 can be determined by alignment with SEQ IDNO:3. Residues corresponding to Y155, R318, R338, T343, R403 and E410are indicated in boxed text.

DETAILED DESCRIPTION

Outline

A. Definitions

B. Hemostasis and Role of Factor IX Therein

-   -   1. Platelet adhesion and aggregation    -   2. Coagulation cascade        -   a. Initiation        -   b. Amplification        -   c. Propagation    -   3. Regulation of Coagulation

C. Factor IX (FIX) Structure and Function

-   -   1. FIX structure    -   2. FIX post-translational modification    -   3. FIX activation    -   4. FIX function    -   5. FIX as a biopharmaceutical

D. Modified FIX polypeptides

-   -   1. Exemplary Amino Acid Replacements        -   a. Altered glycosylation            -   i. Advantages of glycosylation            -   ii. Exemplary modified FIX polypeptides with altered                glycosylation                -   (a). Introduction of non-native glycosylation                    site(s)                -   (b). Elimination of native glycosylation sites        -   b. Increased resistance to AT-III and heparin            -   i. AT-III            -   ii. Heparin            -   iii. Exemplary FIX polypeptides with increased                resistance to AT-III and heparin        -   c. Mutations to increase catalytic activity        -   d. Mutations to decrease LRP binding        -   e. Other mutations to alter posttranslational modification    -   2. Combination modifications        -   a. Modifications to increase activity        -   b. Modifications that increase affinity for phospholipids or            reduce binding to collagen        -   c. Additional modifications to increase resistance to            inhibitors        -   d. Additional modifications to alter glycosylation        -   e. Modifications to increase resistance to proteases        -   f. Modifications to reduce immunogenicity        -   g. Exemplary combination modifications    -   3. Conjugates and fusion proteins

E. Production of FIX polypeptides

-   -   1. Vectors and Cells    -   2. Expression systems        -   a. Prokaryotic expression        -   b. Yeast        -   c. Insects and insect cells        -   d. Mammalian cells        -   e. Plants    -   2. Purification    -   3. Fusion Proteins    -   4. Polypeptide modification    -   5. Nucleotide sequences

F. Assessing modified FIX polypeptide activities

-   -   1. In vitro assays        -   a. Glycosylation        -   b. Other post-translational modifications        -   c. Proteolytic activity        -   d. Coagulation activity        -   e. Binding to and/or inhibition by other proteins and            molecules        -   e. Phospholipid affinity    -   2. Non-human animal models    -   3. Clinical Assays

G. Formulation and Administration

-   -   1. Formulations        -   a. Dosages        -   b. Dosage forms    -   2. Administration of modified FIX polypeptides    -   3. Administration of nucleic acids encoding modified FIX        polypeptides (gene therapy)

H. Therapeutic Uses

-   -   Hemophilia    -   a. Hemophilia B    -   b. Hemophilia A

J. Combination Therapies

K. Articles of manufacture and kits

L. Examples

A. DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which the invention(s) belong. All patents, patent applications,published applications and publications, Genbank sequences, databases,websites and other published materials referred to throughout the entiredisclosure herein, unless noted otherwise, are incorporated by referencein their entirety. In the event that there are a plurality ofdefinitions for terms herein, those in this section prevail. Wherereference is made to a URL or other such identifier or address, it isunderstood that such identifiers can change and particular informationon the internet can come and go, but equivalent information can be foundby searching the internet. Reference thereto evidences the availabilityand public dissemination of such information.

As used herein, coagulation pathway or coagulation cascade refers to theseries of activation events that leads to the formation of an insolublefibrin clot. In the coagulation cascade or pathway, an inactive proteinof a serine protease (also called a zymogen) is converted to an activeprotease by cleavage of one or more peptide bonds, which then serves asthe activating protease for the next zymogen molecule in the cascade. Inthe final proteolytic step of the cascade, fibrinogen is proteolyticallycleaved by thrombin to fibrin, which is then crosslinked at the site ofinjury to form a clot.

As used herein, “hemostasis” refers to the stopping of bleeding or bloodflow in an organ or body part. The term hemostasis can encompass theentire process of blood clotting to prevent blood loss following bloodvessel injury to subsequent dissolution of the blood clot followingtissue repair.

As used herein, “clotting” or “coagulation” refers to the formation ofan insoluble fibrin clot, or the process by which the coagulationfactors of the blood interact in the coagulation cascade, ultimatelyresulting in the formation of an insoluble fibrin clot. As used herein,a “protease” is an enzyme that catalyzes the hydrolysis of covalentpeptidic bonds. These designations include zymogen forms and activatedsingle-, two- and multiple-chain forms thereof. For clarity, referencesto proteases refer to all forms. Proteases include, for example, serineproteases, cysteine proteases, aspartic proteases, threonine andmetallo-proteases depending on the catalytic activity of their activesite and mechanism of cleaving peptide bonds of a target substrate.

As used herein, serine proteases or serine endopeptidases refer to aclass of peptidases, which are characterized by the presence of a serineresidue in the active site of the enzyme. Serine proteases participatein a wide range of functions in the body, including blood clotting andinflammation, as well as functioning as digestive enzymes in prokaryotesand eukaryotes. The mechanism of cleavage by serine proteases is basedon nucleophilic attack of a targeted peptidic bond by a serine.Cysteine, threonine or water molecules associated with aspartate ormetals also can play this role. Aligned side chains of serine, histidineand aspartate form a catalytic triad common to most serine proteases.The active site of serine proteases is shaped as a cleft where thepolypeptide substrate binds.

As used herein, a “factor IX” or FIX polypeptide refers to any factor IXpolypeptide including, but not limited to, a recombinantly producedpolypeptide, a synthetically produced polypeptide and a factor IXpolypeptide extracted or isolated from cells or tissues including, butnot limited to, liver and blood. Alternative names that are usedinterchangeably for factor IX include Factor 9, Christmas factor, plasmathromboplastin component (PTC), coagulation factor IX, and serum factorIX. Abbreviations for factor IX include FIX and F9. Factor IX includesrelated polypeptides from different species including, but not limitedto animals of human and non-human origin. Human factor IX (hFIX)includes factor IX, allelic variant isoforms (such as the allelicvariant having a T148A (SEQ ID NO:20 or 325) or T412P mutation),synthetic molecules from nucleic acids, protein isolated from humantissue and cells, and modified forms thereof. Exemplary unmodifiedmature human factor IX polypeptides include, but are not limited to,unmodified and wild-type native factor IX polypeptides (such as thepolypeptide containing a sequence set forth in SEQ ID NO:3) and theunmodified and wild-type precursor factor IX polypeptide that includes apropeptide and/or a signal peptide (such as, the precursor FIXpolypeptide that has the sequence set forth in SEQ ID NO:2). One ofskill in the art would recognize that the referenced positions of themature factor IX polypeptide (SEQ ID NO:3) differ by 46 amino acidresidues when compared to the precursor FIX polypeptide SEQ ID NO:2,which is the factor IX polypeptide containing the signal peptide andpropeptide sequences Thus, the first amino acid residue of SEQ ID NO:3“corresponds to” the forty-seventh (47^(th)) amino acid residue of SEQID NO:2.

The term “factor IX” also encompasses the activated form of the factorIX polypeptide, called factor IXa (FIXa), containing the FIX light chain(corresponding to amino acids 47-191 of SEQ ID NO:2, and amino acids1-145 of SEQ ID NO:3) and FIX heavy chain (corresponding to amino acids227-461 of SEQ ID NO:2, and amino acids 181-415 of SEQ ID NO:3) linkedby a disulfide bond between residues 132C and 289C (corresponding to themature FIX polypeptide set forth in SEQ ID NO:3). FIXa is produced froma mature FIX polypeptide (e.g. that set forth in SEQ ID NO:3) byproteolytic cleavage after amino acid residues R145 and R180.Proteolytic cleavage can be carried out, for example, by activatedfactor XI (FXIa) or the tissue factor/activated factor VII (TF/FVIIa)complex. The FIX polypeptides provided herein can be further modified,such as by chemical modification or post-translational modification.Such modifications include, but are not limited to, glycosylation,pegylation, albumination, farnysylation, carboxylation, hydroxylation,phosphorylation, and other polypeptide modifications known in the art.

Factor IX includes factor IX from any species, including human andnon-human species. FIX polypeptides of non-human origin include, but arenot limited to, murine, canine, feline, leporine, avian, bovine, ovine,porcine, equine, piscine, ranine, and other primate factor IXpolypeptides. Exemplary FIX polypeptides of non-human origin include,for example, chimpanzee (Pan troglodytes, SEQ ID NO:4), rhesus macaque(Macaca mulatta, SEQ ID NO:5), mouse (Mus musculus, SEQ ID NO:6), rat(Rattus norvegicus, SEQ ID NO:7), Guinea pig (Cavia porcellus, SEQ IDNO:8), pig (Sus scrofa, SEQ ID NO:9), dog (Canis familiaris, SEQ IDNO:10), cat (Felis catus, SEQ ID NO:11), rabbit (Oryctolagus cuniculus,SEQ ID NO:12), chicken (Gallus gallus, SEQ ID NO:13), cow (Bos Taurus,SEQ ID NO:14), sheep (Ovis aries, SEQ ID NO:15), frog (Xenopustropicalis, SEQ ID NO:16), zebrafish (Danio rerio, SEQ ID NO:17), andJapanese pufferfish (Takifugu rubripes, SEQ ID NO:18).

Reference to FIX polypeptides also includes precursor polypeptides andmature FIX polypeptides in single-chain or two-chain forms, truncatedforms thereof that have activity, and includes allelic variants andspecies variants, variants encoded by splice variants, and othervariants, including polypeptides that have at least 40%, 45%, 50%, 55%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity to the precursor polypeptide set forth in SEQ ID NO:2 or themature form thereof (SEQ ID NO:3). Included are modified FIXpolypeptides, such as those of SEQ ID NOS:75-272 and 326-417 andvariants thereof. Also included are those that retain at least anactivity of a FIX, such as FVIIIa binding, factor X binding,phospholipid binding, and/or coagulant activity of a FIX polypeptide. Byretaining activity, the activity can be altered, such as reduced orincreased, as compared to a wild-type FIX so long as the level ofactivity retained is sufficient to yield a detectable effect. FIXpolypeptides include, but are not limited to, tissue-specific isoformsand allelic variants thereof, synthetic molecules prepared bytranslation of nucleic acids, proteins generated by chemical synthesis,such as syntheses that include ligation of shorter polypeptides, throughrecombinant methods, proteins isolated from human and non-human tissueand cells, chimeric FIX polypeptides and modified forms thereof. FIXpolypeptides also include fragments or portions of FIX that are ofsufficient length or include appropriate regions to retain at least oneactivity (upon activation if needed) of a full-length maturepolypeptide. FIX polypeptides also include those that contain chemicalor posttranslational modifications and those that do not containchemical or posttranslational modifications. Such modifications include,but are not limited to, pegylation, albumination, glycosylation,farnysylation, carboxylation, hydroxylation, phosphorylation, and otherpolypeptide modifications known in the art.

As used herein, corresponding residues refers to residues that occur ataligned loci. Related or variant polypeptides are aligned by any methodknown to those of skill in the art. Such methods typically maximizematches, and include methods such as using manual alignments and byusing the numerous alignment programs available (for example, BLASTP)and others known to those of skill in the art. By aligning the sequencesof polypeptides, one skilled in the art can identify correspondingresidues, using conserved and identical amino acid residues as guides.For example, by aligning the sequences of factor IX polypeptides, one ofskill in the art can identify corresponding residues, using conservedand identical amino acid residues as guides. For example, the tyrosinein amino acid position 1 (Y1) of SEQ ID NO:3 (mature factor IX)corresponds to the tyrosine in amino acid position 47 (Y47) of SEQ IDNO:2. In other instances, corresponding regions can be identified. Forexample, the Gla domain corresponds to amino acid positions Y1 throughV46 of SEQ ID NO:3, and to amino acid positions Y47 through V92 of SEQID NO:2. One skilled in the art also can employ conserved amino acidresidues as guides to find corresponding amino acid residues between andamong human and non-human sequences. For example, amino acid residuesQ11 and P74 of SEQ ID NO:3 (human) correspond to R11 and Q74 of SEQ IDNO:14 (bovine). Corresponding positions also can be based on structuralalignments, for example by using computer simulated alignments ofprotein structure. In other instances, corresponding regions can beidentified.

As used herein, the same, with reference to an amino acid replacement,refers to the identical replacement at the reference amino acid positionin SEQ ID NO:3 in a corresponding position in another Factor IXpolypeptide. For example, the same replacement with reference to thereplacement of tyrosine at amino acid residue R318 in SEQ ID NO:3 is thereplacement of tyrosine at amino acid residue R319 in SEQ ID NO:14 (see,for example, FIGS. 3A-3D). For example, the same replacement withreference to the replacement of asparagine at amino acid residue E410 inSEQ ID NO:3 is the replacement of asparagine at amino acid residue S410in SEQ ID NO:366. It is understood that reference to replacement of thesame amino acid refers to replacement of amino acid residues that differat the corresponding position from the replaced residue.

As used herein, a “proregion,” “propeptide,” or “pro sequence,” refersto a region or a segment that is cleaved to produce a mature protein.This can include segments that function to suppress proteolytic activityby masking the catalytic machinery and thus preventing formation of thecatalytic intermediate (i.e., by sterically occluding the substratebinding site). A proregion is a sequence of amino acids positioned atthe amino terminus of a mature biologically active polypeptide and canbe as little as a few amino acids or can be a multidomain structure.

As used herein, “mature factor IX” refers to a FIX polypeptide thatlacks a signal sequence and a propeptide sequence. Typically, a signalsequence targets a protein for secretion via the endoplasmic reticulum(ER)-golgi pathway and is cleaved following insertion into the ER duringtranslation. A propeptide sequence typically functions inpost-translational modification of the protein and is cleaved prior tosecretion of the protein from the cell. Thus, a mature FIX polypeptideis typically a secreted protein. In one example, a mature human FIXpolypeptide is set forth in SEQ ID NO:3. The amino acid sequence setforth in SEQ ID NO:3 differs from that of the precursor polypeptide setforth in SEQ ID NO:2 in that SEQ ID NO:3 is lacking the signal sequence,which corresponds to amino acid residues 1-28 of SEQ ID NO:2, and alsolacks the propeptide sequence, which corresponds to amino acid residues29-46 of SEQ ID NO:2. Reference to a mature FIX polypeptide encompassesthe single-chain zymogen form and the two-chain form. Thus, reference toa mature FIX polypeptide also refers to the two chain form containingthe heavy chain and light chain (without the activation peptidecorresponding to amino acids 192-226 of SEQ ID NO:2) joined by disulfidebonds.

As used herein, “wild-type” or “native” with reference to FIX refers toa FIX polypeptide encoded by a native or naturally occurring FIX gene,including allelic variants, that is present in an organism, including ahuman and other animals, in nature. Reference to wild-type factor IXwithout reference to a species is intended to encompass any species of awild-type factor IX. Included among wild-type FIX polypeptides are theencoded precursor polypeptide, fragments thereof, and processed formsthereof, such as a mature form lacking the signal peptide as well as anypre- or post-translationally processed or modified forms thereof. Alsoincluded among native FIX polypeptides are those that arepost-translationally modified, including, but not limited to,modification by glycosylation, carboxylation and hydroxylation. NativeFIX polypeptides also include single-chain and two-chain forms. Forexample, humans express native FIX. The amino acid sequence of exemplarywild-type human FIX are set forth in SEQ ID NOS:2 and 3 and allelicvariants thereof. Other animals produce native FIX, including, but notlimited to, chimpanzee (Pan troglodytes, SEQ ID NO:4), rhesus macaque(Macaca mulatta, SEQ ID NO:5), mouse (Mus musculus, SEQ ID NO:6), rat(Rattus norvegicus, SEQ ID NO:7), Guinea pig (Cavia porcellus, SEQ IDNO:8), pig (Sus scrofa, SEQ ID NO:9), dog (Canis familiaris, SEQ IDNO:10), cat (Felis catus, SEQ ID NO:11), rabbit (Oryctolagus cuniculus,SEQ ID NO:12), chicken (Gallus gallus, SEQ ID NO:13), cow (Bos Taurus,SEQ ID NO:14), sheep (Ovis aries, SEQ ID NO:15), frog (Xenopustropicalis, SEQ ID NO:16), zebrafish (Danio rerio, SEQ ID NO:17),Japanese pufferfish (Takifugu rubripes, SEQ ID NO:18).

As used herein, species variants refer to variants in polypeptides amongdifferent species, including different mammalian species, such as mouseand human.

As used herein, allelic variants refer to variations in proteins amongmembers of the same species.

As used herein, a splice variant refers to a variant produced bydifferential processing of a primary transcript of genomic DNA thatresults in more than one type of mRNA.

As used herein, a zymogen refers to a protease that is activated byproteolytic cleavage, including maturation cleavage, such as activationcleavage, and/or complex formation with other protein(s) and/orcofactor(s). A zymogen is an inactive precursor of a proteolytic enzyme.Such precursors are generally larger, although not necessarily larger,than the active form. With reference to serine proteases, zymogens areconverted to active enzymes by specific cleavage, including catalyticand autocatalytic cleavage, or by binding of an activating co-factor,which generates an active enzyme. For example, generally, zymogens arepresent in a single-chain form. Zymogens, generally, are inactive andcan be converted to mature active polypeptides by catalytic orautocatalytic cleavage at one or more proteolytic sites to generate amulti-chain, such as a two-chain, polypeptide. A zymogen, thus, is anenzymatically inactive protein that is converted to a proteolytic enzymeby the action of an activator. Cleavage can be effected byautoactivation. A number of coagulation proteins are zymogens; they areinactive, but become cleaved and activated upon the initiation of thecoagulation system following vascular damage. With reference to FIX, theFIX polypeptides exist in the blood plasma as zymogens until cleavage byproteases, such as for example, activated FXI (FXIa) or FVIIa (inassociation with TF) to produce the two-chain form of FIX (FIXa).

As used herein, an activation sequence refers to a sequence of aminoacids in a zymogen that is the site required for activation cleavage ormaturation cleavage to form an active protease. Cleavage of anactivation sequence can be catalyzed autocatalytically or by activatingpartners.

As used herein, activation cleavage is a type of maturation cleavage,which induces a conformation change that is required for the developmentof full enzymatic activity. This is a classical activation pathway, forexample, for serine proteases in which a cleavage generates a newN-terminus that interacts with the conserved regions of the protease,such as Asp 194 in chymotrypsin, to induce conformational changesrequired for activity. Activation can result in production ofmulti-chain forms of the proteases. In some instances, single chainforms of the protease can exhibit proteolytic activity.

As used herein, “activated Factor IX” or “FIXa” refers to any two-chainform of a FIXa polypeptide. A two-chain form typically results fromproteolytic cleavage, but can be produced synthetically. ActivatedFactor IX, thus, includes the zymogen-like two-chain form with lowcoagulant activity, a fully activated form that occurs upon binding toFVIIIa and FX, and mutated forms that exist in a fully activatedtwo-chain form or undergo conformational change to a fully activatedform. For example, a single-chain form of FIX polypeptide (see, e.g.,SEQ ID NO:3) is proteolytically cleaved after amino acid residues R145and R180 of the mature FIX polypeptide. The cleavage products, FIX heavychain and FIX light chain, which are held together by a disulfide bond(between amino acid residues 132C and 289C in the FIX of SEQ ID NO:3),form the two-chain activated FIX enzyme. Proteolytic cleavage can becarried out, for example, by activated factor XIa (FXIa), and activatedfactor VIIa (FVIIa) in complex with TF.

As used herein, a “property” of a FIX polypeptide refers to a physicalor structural property, such three-dimensional structure, pI, half-life,conformation and other such physical characteristics.

As used herein, an “activity” of a FIX polypeptide refers to anyactivity exhibited by a factor IX polypeptide. Such activities can betested in vitro and/or in vivo and include, but are not limited to,coagulation or coagulant activity, pro-coagulant activity, proteolyticor catalytic activity such as to effect factor X (FX) activation;antigenicity (ability to bind to or compete with a polypeptide forbinding to an anti-FIX antibody); ability to bind factor VIIIa or factorX; and/or ability to bind to phospholipids. Activity can be assessed invitro or in vivo using recognized assays, for example, by measuringcoagulation in vitro or in vivo. The results of such assays indicatethat a polypeptide exhibits an activity that can be correlated toactivity of the polypeptide in vivo, in which in vivo activity can bereferred to as biological activity. Assays to determine functionality oractivity of modified forms of FIX are known to those of skill in theart. Exemplary assays to assess the activity of a FIX polypeptideinclude prothromboplastin time (PT) assay or the activated partialthromboplastin time (aPTT) assay to assess coagulant activity, orchromogenic assays using synthetic substrates to assess catalytic orproteolytic activity.

As used herein, “exhibits at least one activity” or “retains at leastone activity” refers to the activity exhibited by a modified FIXpolypeptide as compared to an unmodified FIX polypeptide of the sameform and under the same conditions. For example, a modified FIXpolypeptide in a two-chain form is compared with an unmodified FIXpolypeptide in a two-chain form, under the same experimental conditions,where the only difference between the two polypeptides is themodification under study. In another example, a modified FIX polypeptidein a single-chain form is compared with an unmodified FIX polypeptide ina single-chain form, under the same experimental conditions, where theonly difference between the two polypeptides is the modification understudy. Typically, a modified FIX polypeptide that retains or exhibits atleast one activity of an unmodified FIX polypeptide of the same formretains a sufficient amount of the activity such that, when administeredin vivo, the modified FIX polypeptide is therapeutically effective as aprocoagulant therapeutic. Generally, for a modified FIX polypeptide toretain therapeutic efficacy as a procoagulant, the amount of activitythat is retained is or is about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500% or more of theactivity of an unmodified FIX polypeptide of the same form that displaystherapeutic efficacy as a procoagulant. The amount of activity that isrequired to maintain therapeutic efficacy as a procoagulant can beempirically determined, if necessary. Typically, retention of 0.5% to20%, 0.5% to 10%, 0.5% to 5% of an activity is sufficient to retaintherapeutic efficacy as a procoagulant in vivo.

It is understood that the activity being exhibited or retained by amodified FIX polypeptide can be any activity, including, but not limitedto, coagulation or coagulant activity, pro-coagulant activity;proteolytic or catalytic activity such as to effect factor X (FX)activation; antigenicity (ability to bind to or compete with apolypeptide for binding to an anti-FIX antibody); ability to bind factorVIIIa or factor X; and/or ability to bind to phospholipids. In someinstances, a modified FIX polypeptide can retain an activity that isincreased compared to an unmodified FIX polypeptide. In some cases, amodified FIX polypeptide can retain an activity that is decreasedcompared to an unmodified FIX polypeptide. Activity of a modified FIXpolypeptide can be any level of percentage of activity of the unmodifiedpolypeptide, where both polypeptides are in the same form, including butnot limited to, 1% of the activity, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, 200%, 300%,400%, 500%, or more activity compared to the polypeptide that does notcontain the modification at issue. For example, a modified FIXpolypeptide can exhibit increased or decreased activity compared to theunmodified FIX polypeptide in the same form. For example, it can retainat least about or 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or at least 99% of theactivity of the unmodified FIX polypeptide. In other embodiments, thechange in activity is at least about 2 times, 3 times, 4 times, 5 times,6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 200times, 300 times, 400 times, 500 times, 600 times, 700 times, 800 times,900 times, 1000 times, or more times greater than unmodified FIX. Theparticular level to be retained is a function of the intended use of thepolypeptide and can be empirically determined. Activity can be measured,for example, using in vitro or in vivo assays such as those describedherein.

As used herein, “coagulation activity” or “coagulant activity” or“pro-coagulant activity” refers to the ability of a polypeptide toeffect coagulation. Assays to assess coagulant activity are known tothose of skill in the art, and include prothrombin time (PT) assay orthe activated partial thromboplastin time (aPTT) assay.

As used herein, “catalytic activity” or “proteolytic activity” withreference to FIX refers to the ability of a FIX protein to catalyze theproteolytic cleavage of a substrate, and are used interchangeably.Assays to assess such activities are known in the art. For example, theproteolytic activity of FIX can be measured using chromogenic substratessuch as Mes-D-CHD-Gly-Arg-AMC, where cleavage of the substrate ismonitored by absorbance and the rate of substrate hydrolysis determinedby linear regression.

As used herein, domain (typically a sequence of three or more, generally5 or 7 or more amino acids) refers to a portion of a molecule, such asproteins or the encoding nucleic acids, that is structurally and/orfunctionally distinct from other portions of the molecule and isidentifiable. For example, domains include those portions of apolypeptide chain that can form an independently folded structure withina protein made up of one or more structural motifs and/or that isrecognized by virtue of a functional activity, such as proteolyticactivity. A protein can have one, or more than one, distinct domains.For example, a domain can be identified, defined or distinguished byhomology of the sequence therein to related family members, such ashomology to motifs that define a protease domain or a Gla domain. Inanother example, a domain can be distinguished by its function, such asby proteolytic activity, or an ability to interact with a biomolecule,such as DNA binding, ligand binding, and dimerization. A domainindependently can exhibit a biological function or activity such thatthe domain independently or fused to another molecule can perform anactivity, such as, for example proteolytic activity or ligand binding. Adomain can be a linear sequence of amino acids or a non-linear sequenceof amino acids. Many polypeptides contain a plurality of domains. Suchdomains are known, and can be identified by those of skill in the art.For exemplification herein, definitions are provided, but it isunderstood that it is well within the skill in the art to recognizeparticular domains by name. If needed, appropriate software can beemployed to identify domains.

As used herein, a protease domain is the catalytically active portion ofa protease. Reference to a protease domain of a protease includes thesingle, two- and multi-chain forms of any of these proteins. A proteasedomain of a protein contains all of the requisite properties of thatprotein required for its proteolytic activity, such as for example, thecatalytic center. In reference to FIX, the protease domain shareshomology and structural feature with the chymotrypsin/trypsin familyprotease domains, including the catalytic triad. For example, in themature FIX polypeptide set forth in SEQ ID NO:3, the protease domaincorresponds to amino acid positions 181 to 412.

As used herein, a gamma-carboxyglutamate (Gla) domain refers to theportion of a protein, for example a vitamin K-dependent protein, thatcontains post-translational modifications of glutamate residues,generally most, but not all of the glutamate residues, by vitaminK-dependent carboxylation to form Gla. The Gla domain is responsible forthe high-affinity binding of calcium ions and binding tonegatively-charged phospholipids. Typically, the Gla domain starts atthe N-terminal extremity of the mature form of vitamin K-dependentproteins and ends with a conserved aromatic residue. In a mature FIXpolypeptide the Gla domain corresponds to amino acid positions 1 to 46of the exemplary polypeptide set forth in SEQ ID NO:3. Gla domains arewell known and their locus can be identified in particular polypeptides.The Gla domains of the various vitamin K-dependent proteins sharesequence, structural and functional homology, including the clusteringof N-terminal hydrophobic residues into a hydrophobic patch thatmediates interaction with negatively charged phospholipids on the cellsurface membrane. Exemplary other Gla-containing polypeptides include,but are not limited to, FVII, FX, prothrombin, protein C, protein S,osteocalcin, matrix Gla protein, Growth-arrest-specific protein 6(Gash), and protein Z.

As used herein, an epidermal growth factor (EGF) domain (EGF-1 or EGF-2)refers to the portion of a protein that shares sequence homology to aspecific 30 to 40 amino acid portion of the epidermal growth factor(EGF) sequence. The EGF domain includes six cysteine residues that havebeen shown (in EGF) to be involved in disulfide bonds. The mainstructure of an EGF domain is a two-stranded beta-sheet followed by aloop to a C-terminal short two-stranded sheet. FIX contains two EGFdomains: EGF-1 and EGF-2. These domains correspond to amino acidpositions 47-83, and 84-125, respectively, of the mature FIX polypeptideset forth in SEQ ID NO:3.

As used herein, “unmodified polypeptide” or “unmodified FIX” andgrammatical variations thereof refer to a starting polypeptide that isselected for modification as provided herein. The starting polypeptidecan be a naturally-occurring, wild-type form of a polypeptide. Inaddition, the starting polypeptide can be altered or mutated, such thatit differs from a native wild type isoform but is nonetheless referredto herein as a starting unmodified polypeptide relative to thesubsequently modified polypeptides produced herein. Thus, existingproteins known in the art that have been modified to have a desiredincrease or decrease in a particular activity or property compared to anunmodified reference protein can be selected and used as the startingunmodified polypeptide. For example, a protein that has been modifiedfrom its native form by one or more single amino acid changes andpossesses either an increase or decrease in a desired property, such asa change in a amino acid residue or residues to alter glycosylation, canbe a target protein, referred to herein as unmodified, for furthermodification of either the same or a different property. Exemplarymodified FIX polypeptides known in the art include any FIX polypeptidedescribed in, for example, Schuettrumpf et al., (2005) Blood 105 (6):2316-23; Melton et al., (2001) Blood Coagul. Fibrinolysis 12(4):237-43;Cheung et al., (1992) J. Biol. Chem. 267:20529-20531; Cheung et al.,(1996) Proc. Natl. Acad. Sci. U.S.A. 93:11068-11073; Hopfner et al.,(1997) EMBO J. 16:6626-6635; Sichler et al., (2003) J. Biol. Chem.278:4121-4126; Begbie et al., (2005) Thromb. Haemost. 94(6):1138-47;Chang, J. et al., (1998) J. Biol. Chem. 273(20):12089-94; Yang, L. etal., (2002) J. Biol. Chem. 277(52):50756-60; Yang, L. et al., (2003) J.Biol. Chem. 278(27):25032-8; U.S. Pat. Nos. 5,969,040, 5,621,039,6,423,826, 7,125,841, 6,017,882, 6,531,298; U.S. Patent Publication Nos.20030211094, 20070254840, 20080188414, 2008000422, 20080050772,20080146494, 20080050772, 20080187955, 20040254106, 20050147618,20080280818, 20080102115, 20080167219 and 20080214461; and InternationalPatent Publication Nos. WO2007112005, WO2007135182, WO2008082613,WO2008119815, WO2008119815, WO2007149406, WO2007112005 and WO2004101740.

As used herein, “modified factor IX polypeptides” and “modified factorIX” refer to a FIX polypeptide that has one or more amino aciddifferences compared to an unmodified factor IX polypeptide. The one ormore amino acid differences can be amino acid mutations such as one ormore amino acid replacements (substitutions), insertions or deletions,or can be insertions or deletions of entire domains, and anycombinations thereof. Typically, a modified FIX polypeptide has one ormore modifications in primary sequence compared to an unmodified FIXpolypeptide. For example, a modified FIX polypeptide provided herein canhave 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 30, 40, 50 or more amino acid differences compared to an unmodifiedFIX polypeptide. Any modification is contemplated as long as theresulting polypeptide exhibits at least one FIX activity associated witha native FIX polypeptide, such as, for example, catalytic activity,proteolytic activity, the ability to bind FVIIIa or the ability to bindphospholipids.

As used herein, “antithrombin III” or “AT-III” is a serine proteaseinhibitor (serpin). AT-III is synthesized as a precursor proteincontaining 464 amino acid residues (SEQ ID NO:21) that is cleaved duringsecretion to release a 432 amino acid mature antithrombin (SEQ IDNO:22).

As used herein, “heparin” refers to a heterogeneous group ofstraight-chain highly sulfated glycosaminoglycans having anticoagulantproperties. Heparin can bind to AT-III to form the AT-III/heparincomplex.

As used herein, “increased resistance to AT-III and/or heparin” refersto any amount of decreased sensitivity of a polypeptide, such as amodified FIX polypeptide, to the inhibitory effects of AT-III alone,heparin alone and/or the AT-III/heparin complex compared with areference polypeptide, such as an unmodified FIX polypeptide. Increasedresistance to AT-III, heparin, and/or an AT-III/heparin complex can beassayed by assessing the binding of a modified FIX polypeptide toAT-III, heparin, and/or an AT-III complex. Increased resistance also canbe assayed by measuring inhibition of the catalytic or coagulantactivity of a FIX polypeptide in the presence of AT-III, heparin, or anAT-III/heparin complex. Assays to determine the binding of a polypeptideto an inhibitor or the inhibition of enzymatic activity of a polypeptideby an inhibitor are known in the art. For covalent inhibitors, such as,for example, AT-III or an AT-III/heparin complex, a second order rateconstant for inhibition can be measured. For non-covalent inhibitors,such as, for example, heparin, a k_(i) can be measured. In addition,surface plasma resonance, such as on a BIAcore biosensor instrument,also can be used to measure the binding of FIX polypeptides to AT-III,heparin, and/or an AT-III/heparin complex using one or more definedconditions. However, for covalent inhibitors such as AT-III or anAT-III/heparin complex, only an on-rate can be measured using BIAcore;for non-covalent inhibitors such as heparin, both the on-rate andoff-rate can be measured. Assays to determine the inhibitory effect of,for example, AT-III/heparin on FIX coagulant activity also are known inthe art. For example, the ability of a modified FIX polypeptide tocleave its substrate FX in the presence or absence of AT-III/heparin canbe measured, and the degree to which AT-III/heparin inhibits thereaction determined. This can be compared to the ability of anunmodified FIX polypeptide to cleave its substrate FX in the presence orabsence of AT-III. Alternatively, the second order rate constant forinhibition of a FIX polypeptide can be measured and compared to thesecond order rate constant for inhibition of an unmodified FIXpolypeptide. When comparing second order rate constants for inhibition,increased resistance to inhibition means a decreased second order rateconstant of inhibition. A modified polypeptide that exhibits increasedresistance to AT-III and/or heparin exhibits, for example, an increaseof 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 200%,300%, 400%, 500%, or more resistance to the effects of AT-III, heparin,and/or an AT-III/heparin complex, respectively, compared to anunmodified polypeptide.

As used herein, cofactors refer to proteins or molecules that bind toother specific proteins or molecules to form an active complex. In someexamples, binding to a cofactor is required for optimal proteolyticactivity. For example, FVIIIa is a cofactor of FIXa. Binding of FVIIIato FIXa induces conformational changes that result in increasedproteolytic activity of FIXa for its substrate, FX.

As used herein, a glycosylation site refers to an amino position in apolypeptide to which a carbohydrate moiety can be attached. Typically, aglycosylated protein contains one or more amino acid residues, such asasparagine or serine, for the attachment of the carbohydrate moieties.

As used herein, a native glycosylation site refers to the position of anamino acid to which a carbohydrate moiety is attached in a wild-typepolypeptide. There are six native glycosylation sites in FIX; twoN-glycosylation sites at N157 and N167, and six 0-glycosylation sites atS53, S61, T159, T169, T172 and T179, corresponding to amino acidpositions in the mature FIX polypeptide set forth in SEQ ID NO:3.

As used herein, a non-native glycosylation site refers to the positionof an amino acid to which a carbohydrate moiety is attached in amodified polypeptide that is not present in a wild-type polypeptide.Non-native glycosylation sites can be introduced into a FIX polypeptideby amino acid replacement. O-glycosylation sites can be created, forexample, by amino acid replacement of a native residue with a serine orthreonine. N-glycosylation sites can be created, for example, byestablishing the motif Asn-Xaa-Ser/Thr/Cys, where Xaa is not proline.Creation of this consensus sequence by amino acid modification caninvolve, for example, a single amino acid replacement of a native aminoacid residue with an asparagine, a single amino acid replacement of anative amino acid residue with a serine, threonine or cysteine, or adouble amino acid replacement involving a first amino acid replacementof a native residue with an asparagine and a second amino acidreplacement of native residue with a serine, threonine or cysteine.

As used herein, “increased levels of glycosylation” and any grammaticalvariations thereof, refers to an increased amount of carbohydrate linkedto a polypeptide as compared with a reference polypeptide or protein.The carbohydrate can be N-linked, O-linked, C-linked or be attached byany other linkage. The level of glycosylation can be increased by atleast about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more compared tothe level of glycosylation of an unmodified polypeptide. Assays todetermine the level of glycosylation (i.e. amount of carbohydrate) of apolypeptide are known in the art. For example, the carbohydrate contentor level of glycosylation can be assessed by high pH anion exchangechromatography, fluorophoreassisted carbohydrate electrophoresis (FACE),sequential exoglycosidase digestions, mass spectrometry, NMR, gelelectrophoresis or any other method described herein or known in theart.

As used herein, “biological activity” refers to the in vivo activitiesof a compound or physiological responses that result upon in vivoadministration of a compound, composition or other mixture. Biologicalactivity, thus, encompasses therapeutic effects and pharmaceuticalactivity of such compounds, compositions and mixtures. Biologicalactivities can be observed in in vitro systems designed to test or usesuch activities. Thus, for purposes herein a biological activity of aFIX polypeptide encompasses the coagulant activity.

As used herein, a pharmacokinetic property refers to a property relatedto the action of a drug or agent, such as a FIX polypeptide, in the bodyand in particular the rate at which drugs are absorbed, distributed,metabolized, and eliminated by the body. Pharmacokinetics can beassessed by various parameters. These include, but are not limited to,clearance, volume of distribution, in vivo recovery, total modified FIXpolypeptide exposure in vivo, serum half-life, and mean resonance time(MRT). Pharmacokinetic properties of polypeptide can be assessed usingmethods well known in the art, such as, for example, administering thepolypeptide to a human or animal model and assessing the amount of FIXin the body at various time points. The various parameters, such asclearance, volume of distribution, in vivo recovery, total modified FIXpolypeptide exposure in vivo, serum half-life, and mean resonance time(MRT), are assessed using calculations well known in the art anddescribed herein.

As used herein, “improved pharmacokinetic properties” refers to adesirable change in a pharmacokinetic property of a polypeptide, such asa modified FIX polypeptide, compared to, for example, an unmodified FIXpolypeptide. The change can be an increase or a decrease.

As used herein, clearance refers to the removal of an agent, such as apolypeptide, from the body of a subject following administration.Clearance can be assessed using methods well known in the art, such asthose described in Example 6. For example, assays in which a FIXpolypeptide is administered to mice can be performed, and the clearanceof the polypeptide from the body assessed by measuring the amount of FIXin the plasma at various time points and calculating the clearance asDose/AUC_(0-inf). Improved clearance of a modified FIX polypeptidecompared to an unmodified FIX polypeptide refers to a decrease inclearance of a modified FIX polypeptide compared to an unmodified FIXpolypeptide. The clearance of a modified FIX polypeptide can bedecreased by at least or about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% compared to an unmodified FIX polypeptide.

As used herein, mean resonance time (MRT) refers to the amount of time aFIX polypeptide resides in the body following administration. MRT can beassessed using methods well known in the art, such as those described inExample 6. For example, assays in which a FIX polypeptide isadministered to mice can be performed, and the MRT of the polypeptideassessed by measuring the amount of FIX in the plasma at various timepoints and calculating the MRT as AUMC_(0-last)/AUC_(0-last), whereAUC_(0-last) is total area under the curve and AUMC_(0-last) is thetotal area under the first moment-versus-time curve. Improved MRT of amodified FIX polypeptide compared to an unmodified FIX polypeptiderefers to an increase in MRT of a modified FIX polypeptide compared toan unmodified FIX polypeptide. The MRT of a modified FIX polypeptide canbe increased by at least or about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, 100%, 200%, 300%, 400%, 500% or more compared to anunmodified FIX polypeptide.

As used herein, in vivo recovery refers to the percentage of FIXpolypeptide detectable in the circulation after a period of timefollowing administration in relation to the total amount of FIXpolypeptide administered. In vivo recovery can be assessed using methodswell known in the art, such as those described in Example 6. Forexample, assays in which a FIX polypeptide is administered to mice canbe performed, and the in vivo recovery of the polypeptide assessed bymeasuring the amount of FIX in the plasma at C_(max) and comparing it tothe amount of FIX administered. Improved in vivo recovery of a modifiedFIX polypeptide compared to an unmodified FIX polypeptide refers to anincrease in in vivo recovery of a modified FIX polypeptide compared toan unmodified FIX polypeptide. The in vivo recovery of a modified FIXpolypeptide can be increased by at least or about 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 200%, 300%, 400%, 500% or morecompared to an unmodified FIX polypeptide.

As used herein, plasma half-life (t_(1/2)) refers the eliminationhalf-life of a FIX polypeptide, or the time at which the plasmaconcentration of the FIX polypeptide has reached one half of its initialor maximal concentration following administration. Reference to plasmahalf-life includes plasma half-life during the α-, β-, and/or γ-phase.Plasma half-life can be assessed using methods well known in the art,such as those described in Example 6. For example, assays in which a FIXpolypeptide is administered to mice can be performed, and the plasmahalf-life of the polypeptide assessed by measuring the amount of FIX inthe plasma at various time points. The T_(1/2β), for example, iscalculated as −ln 2 divided by the negative slope during the terminalphase of the log-linear plot of the plasma FIX concentration-versus-timecurve. Improved plasma half-life of a modified FIX polypeptide comparedto an unmodified FIX polypeptide refers to an increase in plasmahalf-life of a modified FIX polypeptide compared to an unmodified FIXpolypeptide. The plasma half-life of a modified FIX polypeptide can beincreased by at least or about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 100%, 200%, 300%, 400%, 500% or more compared to anunmodified FIX polypeptide.

As used herein, exposure in vivo refers to the amount of FIX polypeptidein the circulation following administration in relation to the plasmaarea under the concentration-time curve, or AUC, of FIX polypeptideadministered. Exposure in vivo can be assessed using methods well knownin the art, such as those described in Example 6. For example, assays inwhich a FIX polypeptide is administered to mice can be performed, andthe in vivo recovery of the polypeptide assessed by measuring the amountof FIX in the plasma at various time points (i.e., AUC) and comparing itto the amount of FIX administered. Improved exposure in vivo of amodified FIX polypeptide compared to an unmodified FIX polypeptiderefers to an increase in exposure in vivo of a modified FIX polypeptidecompared to an unmodified FIX polypeptide. The exposure in vivo of amodified FIX polypeptide can be increased by at least or about 1%, 2%,3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 200%, 300%, 400%,500% or more compared to an unmodified FIX polypeptide.

As used herein, volume of distribution refers to the distribution of aFIX polypeptide between plasma and the rest of the body followingadministration. It is defined as the volume in which the amount ofpolypeptide would need to be uniformly distributed to produce theobserved concentration of polypeptide in the plasma. Volume ofdistribution can be assessed using methods well known in the art, suchas those described in Example 6. For example, V_(ss), which is thesteady state volume of distribution (calculated as MRT*Cl) and V_(z),which is the volume of distribution based on the terminal eliminationconstant (β) (calculated as Cl/(ln 2/T_(1/2β)), can be assessed inassays in which a FIX polypeptide is administered to mice, and theconcentration of the FIX in the plasma is determined at various timepoints. Improved volume of distribution of a modified FIX polypeptidecompared with an unmodified FIX polypeptide, depending on the protein'smechanism of clearance and safety profile, can refer to either anincrease or a decrease in the volume of distribution of a modified FIXpolypeptide. For example, in cases where the polypeptide is distributedamong multiple compartments, a decreased volume of distribution of amodified FIX polypeptide could result in significantly increased drugexposure and activity in the compartment of interest (e.g., the vascularcompartment versus an extravascular compartment) compared with anunmodified FIX polypeptide. In other cases, for example, when drugsafety is limited by C_(max), redistribution into other compartments(e.g., binding to the surface of endothelial cells) can result in alonger terminal half life and/or duration of action within thecompartment of interest and an superior safety profile compared to theunmodified FIX polypeptide. The volume of distribution of a modified FIXpolypeptide can be decreased by at least or about 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% compared to an unmodified FIXpolypeptide. In other examples, the volume of distribution of themodified FIX polypeptide is increased by at least or about 1%, 2%, 3%,4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 200%, 300%, 400%,500% or more of the volume of distribution of an unmodified FIXpolypeptide.

As used herein the term “assess”, and grammatical variations thereof, isintended to include quantitative and qualitative determination in thesense of obtaining an absolute value for the activity of a polypeptide,and also of obtaining an index, ratio, percentage, visual or other valueindicative of the level of the activity. Assessment can be direct orindirect. For example, detection of cleavage of a substrate by apolypeptide can be by direct measurement of the product, or can beindirectly measured by determining the resulting activity of the cleavedsubstrate.

As used herein, “chymotrypsin numbering” refers to the amino acidnumbering of a mature bovine chymotrypsin polypeptide of SEQ ID NO:19.Alignment of a protease domain of another protease, such as for examplethe protease domain of Factor IX, can be made with chymotrypsin. In suchan instance, the amino acids of Factor IX that correspond to amino acidsof chymotrypsin are given the numbering of the chymotrypsin amino acids.Corresponding positions can be determined by such alignment by one ofskill in the art using manual alignments or by using the numerousalignment programs available (for example, BLASTP). Correspondingpositions also can be based on structural alignments, for example byusing computer simulated alignments of protein structure. Recitationthat amino acids of a polypeptide correspond to amino acids in adisclosed sequence refers to amino acids identified upon alignment ofthe polypeptide with the disclosed sequence to maximize identity orhomology (where conserved amino acids are aligned) using a standardalignment algorithm, such as the GAP algorithm. The correspondingchymotrypsin numbers of amino acid positions 181 to 415 of the FIXpolypeptide set forth in SEQ ID NO:3 are provided in Table 1. The aminoacid positions relative to the sequence set forth in SEQ ID NO:3 are innormal font, the amino acid residues at those positions are in bold, andthe corresponding chymotrypsin numbers are in italics. For example, uponalignment of the mature Factor IX (SEQ ID NO:3) with mature chymotrypsin(SEQ ID NO:19), the valine (V) at amino acid position 181 in Factor IXis given the chymotrypsin numbering of V16. Subsequent amino acids arenumbered accordingly. In one example, a glutamic acid (E) at amino acidposition 213 of the mature factor IX (SEQ ID NO:3) corresponds to aminoacid position E49 based on chymotrypsin numbering. Where a residueexists in a protease, but is not present in chymotrypsin, the amino acidresidue is given a letter notation. For example, A95a and A95b bychymotrypsin numbering correspond to A261 and A262, respectively, bynumbering relative to the mature Factor IX sequence (SEQ ID NO:3).

TABLE 1 Chymotrypsin numbering of Factor IX 181 182 183 184 185 186 187188 189 190 191 192 193 194 195 V V G G E D A K P G Q F P W Q  16  17 18  19  20  21  22  23  24  25  26  27  28  29  30 196 197 198 199 200201 202 203 204 205 206 207 208 209 210 V V L N G K V D A F C G G S I 31  32  33  34  35  37  38  39  40  41  42  43  44  45  46 211 212 213214 215 216 217 218 219 220 221 222 223 224 225 V N E K W I V T A A H CV E T  47  48  49  50  51  52  53  54  55  56  57  58  59  60   60A 226227 228 229 230 231 232 233 234 235 236 237 238 239 240 G V K I T V V AG E H N I E E  61  62  63  64  65  66  67  68  69  70  71  72  73  74 75 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 T E H TE Q K R N V I R I I P  76  77  78  79  80  81  82  83  84  85  86  87 88  89  90 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270H H N Y N A A I N K Y N H D I  91  92  93  94  95   95A   95B  96  97 98  99 100 101 102 103 271 272 273 274 275 276 277 278 279 280 281 282283 284 285 A L L E L D E P L V L N S Y V 104 105 106 107 108 109 110111 112 113 114 115 116 117 118 286 287 288 289 290 291 292 293 294 295296 297 298 299 300 T P I C I A D K E Y T N I F L 119 120 121 122 123124 125 126 127 128 129   129A   129B 130 131 301 302 303 304 305 306307 308 309 310 311 312 313 314 315 K F G S G Y V S G W G R V F H 132133 134 135 136 137 138 139 140 141 142 143 144 145 147 316 317 318 319320 321 322 323 324 325 326 327 328 329 330 K G R S A L V L Q Y L R V PL 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 331 332333 334 335 336 337 338 339 340 341 342 343 344 345 V D R A T C L R S TK F T I Y 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 N N M F C AG F H E G G R D S 178 179 180 181 182 183 184   184A 185 186 187 188  188A 189 190 361 362 363 364 365 366 367 368 369 370 371 372 373 374375 C Q G D S G G P H V T E V E G 191 192 193 194 195 196 197 198 199200 201 202 203 204 205 376 377 378 379 380 381 382 383 384 385 386 387388 389 390 T S F L T G I I S W G E E C A 206 207 208 209 210 211 212213 214 215 216 217 219 220 221 391 392 393 394 395 396 397 398 399 400401 402 403 404 405 M K G K Y G I Y T K V S R Y V   221A 222 223 224 225226 227 228 229 230 231 232 233 234 235 406 407 408 409 410 411 412 413414 415 N W I K E K T K L T 236 237 328 239 240 241 242 243 244 245

As used herein, nucleic acids include DNA, RNA and analogs thereof,including peptide nucleic acids (PNA) and mixtures thereof. Nucleicacids can be single or double-stranded. When referring to probes orprimers, which are optionally labeled, such as with a detectable label,such as a fluorescent or radiolabel, single-stranded molecules arecontemplated. Such molecules are typically of a length such that theirtarget is statistically unique or of low copy number (typically lessthan 5, generally less than 3) for probing or priming a library.Generally a probe or primer contains at least 14, 16 or 30 contiguousnucleotides of sequence complementary to or identical to a gene ofinterest. Probes and primers can be 10, 20, 30, 50, 100 or morenucleotides long.

As used herein, a peptide refers to a polypeptide that is from 2 to 40amino acids in length.

As used herein, the amino acids that occur in the various sequences ofamino acids provided herein are identified according to their known,three-letter or one-letter abbreviations (Table 2). The nucleotideswhich occur in the various nucleic acid fragments are designated withthe standard single-letter designations used routinely in the art.

As used herein, an “amino acid” is an organic compound containing anamino group and a carboxylic acid group. A polypeptide contains two ormore amino acids. For purposes herein, amino acids include the twentynaturally-occurring amino acids, non-natural amino acids and amino acidanalogs (i.e., amino acids wherein the α-carbon has a side chain).

In keeping with standard polypeptide nomenclature described in J. Biol.Chem., 243:3557-3559 (1968), and adopted in 37 C.F.R. §§1.821-1.822,abbreviations for the amino acid residues are shown in Table 2A:

TABLE 2A Table of Correspondence SYMBOL 1-Letter 3-Letter AMINO ACID YTyr Tyrosine G Gly Glycine F Phe Phenylalanine M Met Methionine A AlaAlanine S Ser Serine I Ile Isoleucine L Leu Leucine T Thr Threonine VVal Valine P Pro Proline K Lys Lysine H His Histidine Q Gln Glutamine EGlu Glutamic acid Z Glx Glu and/or Gln W Trp Tryptophan R Arg Arginine DAsp Aspartic acid N Asn Asparagine B Asx Asn and/or Asp C Cys Cysteine XXaa Unknown or other

It should be noted that all amino acid residue sequences representedherein by formulae have a left to right orientation in the conventionaldirection of amino-terminus to carboxyl-terminus. In addition, thephrase “amino acid residue” is broadly defined to include the aminoacids listed in the Table of Correspondence (Table 2) and modified andunusual amino acids, such as those referred to in 37 C.F.R.§§1.821-1.822, and incorporated herein by reference. Furthermore, itshould be noted that a dash at the beginning or end of an amino acidresidue sequence indicates a peptide bond to a further sequence of oneor more amino acid residues, to an amino-terminal group such as NH₂ orto a carboxyl-terminal group such as COOH.

As used herein, “naturally occurring amino acids” refer to the 20L-amino acids that occur in polypeptides.

As used herein, “non-natural amino acid” refers to an organic compoundcontaining an amino group and a carboxylic acid group that is not one ofthe naturally-occurring amino acids listed in Table 2. Non-naturallyoccurring amino acids thus include, for example, amino acids or analogsof amino acids other than the 20 naturally-occurring amino acids andinclude, but are not limited to, the D-isostereomers of amino acids.Exemplary non-natural amino acids are known to those of skill in the artand can be included in a modified Factor IX polypeptide.

For purposes herein, conservative amino acid substitutions may be madein any of polypeptides and domains thereof provided that the resultingprotein exhibits an activity of a FIX. Conservative amino acidsubstitutions, such as those set forth in Table 2B, are those that donot eliminate proteolytic activity. Suitable conservative substitutionsof amino acids are known to those of skill in this art and may be madegenerally without altering the biological activity of the resultingmolecule. Those of skill in this art recognize that, in general, singleamino acid substitutions in non-essential regions of a polypeptide donot substantially alter biological activity (see, e.g., Watson et al.Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin/CummingsPub. co., p. 224). Also included within the definition, is thecatalytically active fragment of an MTSP, particularly a single chainprotease portion. Conservative amino acid substitutions are made, forexample, in accordance with those set forth in Table 2B as follows:

TABLE 2B Original residue Conservative substitution Ala (A) Gly; Ser,Abu Arg (R) Lys, orn Asn (N) Gln; His Cys (C) Ser Gln (Q) Asn Glu (E)Asp Gly (G) Ala; Pro His (H) Asn; Gln Ile (I) Leu; Val; Met; Nle; NvaLeu (L) Ile; Val; Met; Nle; Nv Lys (K) Arg; Gln; Glu Met (M) Leu; Tyr;Ile; NLe Val Ornithine Lys; Arg Phe (F) Met; Leu; Tyr Ser (S) Thr Thr(T) Ser Trp (W) Tyr Tyr (Y) Trp; Phe Val (V) Ile; Leu; Met; Nle; NvOther substitutions are also permissible and may be determinedempirically or in accord with known conservative substitutions.

As used herein, a DNA construct is a single or double stranded, linearor circular DNA molecule that contains segments of DNA combined andjuxtaposed in a manner not found in nature. DNA constructs exist as aresult of human manipulation, and include clones and other copies ofmanipulated molecules.

As used herein, a DNA segment is a portion of a larger DNA moleculehaving specified attributes. For example, a DNA segment encoding aspecified polypeptide is a portion of a longer DNA molecule, such as aplasmid or plasmid fragment, which, when read from the 5′ to 3′direction, encodes the sequence of amino acids of the specifiedpolypeptide.

As used herein, the term polynucleotide means a single- ordouble-stranded polymer of deoxyribonucleotides or ribonucleotide basesread from the 5′ to the 3′ end. Polynucleotides include RNA and DNA, andcan be isolated from natural sources, synthesized in vitro, or preparedfrom a combination of natural and synthetic molecules. The length of apolynucleotide molecule is given herein in terms of nucleotides(abbreviated “nt”) or base pairs (abbreviated “bp”). The termnucleotides is used for single- and double-stranded molecules where thecontext permits. When the term is applied to double-stranded moleculesit is used to denote overall length and will be understood to beequivalent to the term base pairs. It will be recognized by thoseskilled in the art that the two strands of a double-strandedpolynucleotide can differ slightly in length and that the ends thereofcan be staggered; thus all nucleotides within a double-strandedpolynucleotide molecule can not be paired. Such unpaired ends will, ingeneral, not exceed 20 nucleotides in length.

As used herein, “primary sequence” refers to the sequence of amino acidresidues in a polypeptide.

As used herein, “similarity” between two proteins or nucleic acidsrefers to the relatedness between the sequence of amino acids of theproteins or the nucleotide sequences of the nucleic acids. Similaritycan be based on the degree of identity and/or homology of sequences ofresidues and the residues contained therein. Methods for assessing thedegree of similarity between proteins or nucleic acids are known tothose of skill in the art. For example, in one method of assessingsequence similarity, two amino acid or nucleotide sequences are alignedin a manner that yields a maximal level of identity between thesequences. “Identity” refers to the extent to which the amino acid ornucleotide sequences are invariant. Alignment of amino acid sequences,and to some extent nucleotide sequences, also can take into accountconservative differences and/or frequent substitutions in amino acids(or nucleotides). Conservative differences are those that preserve thephysico-chemical properties of the residues involved. Alignments can beglobal (alignment of the compared sequences over the entire length ofthe sequences and including all residues) or local (the alignment of aportion of the sequences that includes only the most similar region orregions).

As used herein, the terms “homology” and “identity” are usedinterchangeably, but homology for proteins can include conservativeamino acid changes. In general to identify corresponding positions thesequences of amino acids are aligned so that the highest order match isobtained (see, e.g.: Computational Molecular Biology, Lesk, A. M., ed.,Oxford University Press, New York, 1988; Biocomputing: Informatics andGenome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991; Carillo et al. (1988) SIAM J Applied Math 48:1073).

As use herein, “sequence identity” refers to the number of identicalamino acids (or nucleotide bases) in a comparison between a test and areference polypeptide or polynucleotide. Homologous polypeptides referto a pre-determined number of identical or homologous amino acidresidues. Homology includes conservative amino acid substitutions aswell identical residues. Sequence identity can be determined by standardalignment algorithm programs used with default gap penalties establishedby each supplier. Homologous nucleic acid molecules refer to apre-determined number of identical or homologous nucleotides. Homologyincludes substitutions that do not change the encoded amino acid (i.e.,“silent substitutions”) as well identical residues. Substantiallyhomologous nucleic acid molecules hybridize typically at moderatestringency or at high stringency all along the length of the nucleicacid or along at least about 70%, 80% or 90% of the full-length nucleicacid molecule of interest. Also contemplated are nucleic acid moleculesthat contain degenerate codons in place of codons in the hybridizingnucleic acid molecule. (For determination of homology of proteins,conservative amino acids can be aligned as well as identical aminoacids; in this case, percentage of identity and percentage homologyvaries). Whether any two nucleic acid molecules have nucleotidesequences (or any two polypeptides have amino acid sequences) that areat least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% “identical” can bedetermined using known computer algorithms such as the “FAST A” program,using for example, the default parameters as in Pearson et al. Proc.Natl. Acad. Sci. USA 85: 2444 (1988) (other programs include the GCGprogram package (Devereux, J., et al., Nucleic Acids Research 12(I): 387(1984)), BLASTP, BLASTN, FASTA (Atschul, S. F., et al., J. Molec. Biol.215:403 (1990); Guide to Huge Computers, Martin J. Bishop, ed., AcademicPress, San Diego (1994), and Carillo et al. SIAM J Applied Math 48: 1073(1988)). For example, the BLAST function of the National Center forBiotechnology Information database can be used to determine identity.Other commercially or publicly available programs include DNAStar“MegAlign” program (Madison, Wis.) and the University of WisconsinGenetics Computer Group (UWG) “Gap” program (Madison Wis.)). Percenthomology or identity of proteins and/or nucleic acid molecules can bedetermined, for example, by comparing sequence information using a GAPcomputer program (e.g., Needleman et al. J. Mol. Biol. 48: 443 (1970),as revised by Smith and Waterman (Adv. Appl. Math. 2: 482 (1981)).Briefly, a GAP program defines similarity as the number of alignedsymbols (i.e., nucleotides or amino acids) which are similar, divided bythe total number of symbols in the shorter of the two sequences. Defaultparameters for the GAP program can include: (1) a unary comparisonmatrix (containing a value of 1 for identities and 0 for non identities)and the weighted comparison matrix of Gribskov et al. Nucl. Acids Res.14: 6745 (1986), as described by Schwartz and Dayhoff, eds., Atlas ofProtein Sequence and Structure, National Biomedical Research Foundation,pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and an additional0.10 penalty for each symbol in each gap; and (3) no penalty for endgaps.

Therefore, as used herein, the term “identity” represents a comparisonbetween a test and a reference polypeptide or polynucleotide. In onenon-limiting example, “at least 90% identical to” refers to percentidentities from 90 to 100% relative to the reference polypeptides.Identity at a level of 90% or more is indicative of the fact that,assuming for exemplification purposes a test and referencepolynucleotide length of 100 amino acids are compared, no more than 10%(i.e., 10 out of 100) of amino acids in the test polypeptide differ fromthat of the reference polypeptides. Similar comparisons can be madebetween test and reference polynucleotides. Such differences can berepresented as point mutations randomly distributed over the entirelength of an amino acid sequence or they can be clustered in one or morelocations of varying length up to the maximum allowable, e.g., 10/100amino acid difference (approximately 90% identity). Differences aredefined as nucleic acid or amino acid substitutions, insertions ordeletions. At the level of homologies or identities above about 85-90%,the result should be independent of the program and gap parameters set;such high levels of identity can be assessed readily, often withoutrelying on software.

As used herein, it also is understood that the terms “substantiallyidentical” or “similar” varies with the context as understood by thoseskilled in the relevant art, but that those of skill can assess such.

As used herein, an aligned sequence refers to the use of homology(similarity and/or identity) to align corresponding positions in asequence of nucleotides or amino acids. Typically, two or more sequencesthat are related by 50% or more identity are aligned. An aligned set ofsequences refers to 2 or more sequences that are aligned atcorresponding positions and can include aligning sequences derived fromRNAs, such as ESTs and other cDNAs, aligned with genomic DNA sequence.

As used herein, “specifically hybridizes” refers to annealing, bycomplementary base-pairing, of a nucleic acid molecule (e.g. anoligonucleotide) to a target nucleic acid molecule. Those of skill inthe art are familiar with in vitro and in vivo parameters that affectspecific hybridization, such as length and composition of the particularmolecule. Parameters particularly relevant to in vitro hybridizationfurther include annealing and washing temperature, buffer compositionand salt concentration. Exemplary washing conditions for removingnon-specifically bound nucleic acid molecules at high stringency are0.1×SSPE, 0.1% SDS, 65° C., and at medium stringency are 0.2×SSPE, 0.1%SDS, 50° C. Equivalent stringency conditions are known in the art. Theskilled person can readily adjust these parameters to achieve specifichybridization of a nucleic acid molecule to a target nucleic acidmolecule appropriate for a particular application.

As used herein, isolated or purified polypeptide or protein orbiologically-active portion thereof is substantially free of cellularmaterial or other contaminating proteins from the cell of tissue fromwhich the protein is derived, or substantially free from chemicalprecursors or other chemicals when chemically synthesized. Preparationscan be determined to be substantially free if they appear free ofreadily detectable impurities as determined by standard methods ofanalysis, such as thin layer chromatography (TLC), gel electrophoresisand high performance liquid chromatography (HPLC), used by those ofskill in the art to assess such purity, or sufficiently pure such thatfurther purification would not detectably alter the physical andchemical properties, such as proteolytic and biological activities, ofthe substance. Methods for purification of the compounds to producesubstantially chemically pure compounds are known to those of skill inthe art. A substantially chemically pure compound, however, can be amixture of stereoisomers. In such instances, further purification mightincrease the specific activity of the compound.

The term substantially free of cellular material includes preparationsof proteins in which the protein is separated from cellular componentsof the cells from which it is isolated or recombinantly-produced. In oneembodiment, the term substantially free of cellular material includespreparations of protease proteins having less that about 30% (by dryweight) of non-protease proteins (also referred to herein as acontaminating protein), generally less than about 20% of non-proteaseproteins or 10% of non-protease proteins or less that about 5% ofnon-protease proteins. When the protease protein or active portionthereof is recombinantly produced, it also is substantially free ofculture medium, i.e., culture medium represents less than, about, orequal to 20%, 10% or 5% of the volume of the protease proteinpreparation.

As used herein, the term substantially free of chemical precursors orother chemicals includes preparations of protease proteins in which theprotein is separated from chemical precursors or other chemicals thatare involved in the synthesis of the protein. The term includespreparations of protease proteins having less than about 30% (by dryweight), 20%, 10%, 5% or less of chemical precursors or non-proteasechemicals or components.

As used herein, production by recombinant methods by using recombinantDNA methods refers to the use of the well known methods of molecularbiology for expressing proteins encoded by cloned DNA.

As used herein, vector (or plasmid) refers to discrete elements that areused to introduce heterologous nucleic acid into cells for eitherexpression or replication thereof. The vectors typically remainepisomal, but can be designed to effect integration of a gene or portionthereof into a chromosome of the genome. Also contemplated are vectorsthat are artificial chromosomes, such as bacterial artificialchromosomes, yeast artificial chromosomes and mammalian artificialchromosomes. Selection and use of such vehicles are well known to thoseof skill in the art.

As used herein, expression refers to the process by which nucleic acidis transcribed into mRNA and translated into peptides, polypeptides, orproteins. If the nucleic acid is derived from genomic DNA, expressioncan, if an appropriate eukaryotic host cell or organism is selected,include processing, such as splicing of the mRNA.

As used herein, an expression vector includes vectors capable ofexpressing DNA that is operatively linked with regulatory sequences,such as promoter regions, that are capable of effecting expression ofsuch DNA fragments. Such additional segments can include promoter andterminator sequences, and optionally can include one or more origins ofreplication, one or more selectable markers, an enhancer, apolyadenylation signal, and the like. Expression vectors are generallyderived from plasmid or viral DNA, or can contain elements of both.Thus, an expression vector refers to a recombinant DNA or RNA construct,such as a plasmid, a phage, recombinant virus or other vector that, uponintroduction into an appropriate host cell, results in expression of thecloned DNA. Appropriate expression vectors are well known to those ofskill in the art and include those that are replicable in eukaryoticcells and/or prokaryotic cells and those that remain episomal or thosewhich integrate into the host cell genome.

As used herein, vector also includes “virus vectors” or “viral vectors.”Viral vectors are engineered viruses that are operatively linked toexogenous genes to transfer (as vehicles or shuttles) the exogenousgenes into cells.

As used herein, an adenovirus refers to any of a group of DNA-containingviruses that cause conjunctivitis and upper respiratory tract infectionsin humans.

As used herein, naked DNA refers to histone-free DNA that can be usedfor vaccines and gene therapy. Naked DNA is the genetic material that ispassed from cell to cell during a gene transfer processed calledtransformation or transfection. In transformation or transfection,purified or naked DNA that is taken up by the recipient cell will givethe recipient cell a new characteristic or phenotype.

As used herein, operably or operatively linked when referring to DNAsegments means that the segments are arranged so that they function inconcert for their intended purposes, e.g., transcription initiates inthe promoter and proceeds through the coding segment to the terminator.

As used herein, an agent that modulates the activity of a protein orexpression of a gene or nucleic acid either decreases or increases orotherwise alters the activity of the protein or, in some manner, up- ordown-regulates or otherwise alters expression of the nucleic acid in acell.

As used herein, a “chimeric protein” or “fusion protein” refers to apolypeptide operatively-linked to a different polypeptide. A chimeric orfusion protein provided herein can include one or more FIX polypeptides,or a portion thereof, and one or more other polypeptides for any one ormore of a transcriptional/translational control signals, signalsequences, a tag for localization, a tag for purification, part of adomain of an immunoglobulin G, and/or a targeting agent. A chimeric FIXpolypeptide also includes those having their endogenous domains orregions of the polypeptide exchanged with another polypeptide. Thesechimeric or fusion proteins include those produced by recombinant meansas fusion proteins, those produced by chemical means, such as bychemical coupling, through, for example, coupling to sulfhydryl groups,and those produced by any other method whereby at least one polypeptide(i.e. FIX), or a portion thereof, is linked, directly or indirectly vialinker(s) to another polypeptide.

As used herein, operatively-linked when referring to a fusion proteinrefers to a protease polypeptide and a non-protease polypeptide that arefused in-frame to one another. The non-protease polypeptide can be fusedto the N-terminus or C-terminus of the protease polypeptide.

As used herein, a targeting agent, is any moiety, such as a protein oreffective portion thereof, that provides specific binding to a cellsurface molecule, such a cell surface receptor, which in some instancescan internalize a bound conjugate or portion thereof. A targeting agentalso can be one that promotes or facilitates, for example, affinityisolation or purification of the conjugate; attachment of the conjugateto a surface; or detection of the conjugate or complexes containing theconjugate.

As used herein, derivative or analog of a molecule refers to a portionderived from or a modified version of the molecule.

As used herein, “disease or disorder” refers to a pathological conditionin an organism resulting from cause or condition including, but notlimited to, infections, acquired conditions, genetic conditions, andcharacterized by identifiable symptoms. Diseases and disorders ofinterest herein are those involving coagulation, including thosemediated by coagulation proteins and those in which coagulation proteinsplay a role in the etiology or pathology. Diseases and disorders alsoinclude those that are caused by the absence of a protein such as inhemophilia, and of particular interest herein are those disorders wherecoagulation does not occur due to a deficiency of defect in acoagulation protein.

As used herein, “procoagulant” refers to any substance that promotesblood coagulation.

As used herein, “anticoagulant” refers to any substance that inhibitsblood coagulation

As used herein, “hemophilia” refers to a bleeding disorder caused by adeficiency in blood clotting factors. Hemophilia can be the result, forexample, of absence, reduced expression, or reduced function of aclotting factor. The most common type of hemophilia is hemophilia A,which results from a deficiency in factor VIII. The second most commontype of hemophilia is hemophilia B, which results from a deficiency infactor IX. Hemophilia C, also called FXI deficiency, is a milder andless common form of hemophilia.

As used herein, “congenital hemophilia” refers to types of hemophiliathat are inherited. Congenital hemophilia results from mutation,deletion, insertion, or other modification of a clotting factor gene inwhich the production of the clotting factor is absent, reduced, ornon-functional. For example, hereditary mutations in clotting factorgenes, such as factor VIII and factor IX result in the congenitalhemophilias, Hemophilia A and B, respectively.

As used herein, “acquired hemophilia” refers to a type of hemophiliathat develops in adulthood from the production of autoantibodies thatinactivate FVIII.

As used herein, “bleeding disorder” refers to a condition in which thesubject has a decreased ability to control bleeding. Bleeding disorderscan be inherited or acquired, and can result from, for example, defectsor deficiencies in the coagulation pathway, defects or deficiencies inplatelet activity, or vascular defects.

As used herein, “acquired bleeding disorder” refers to bleedingdisorders that result from clotting deficiencies caused by conditionssuch as liver disease, vitamin K deficiency, or coumadin (warfarin) orother anti-coagulant therapy.

As used herein, “treating” a subject having a disease or condition meansthat a polypeptide, composition or other product provided herein isadministered to the subject.

As used herein, a therapeutic agent, therapeutic regimen,radioprotectant, or chemotherapeutic mean conventional drugs and drugtherapies, including vaccines, which are known to those skilled in theart. Radiotherapeutic agents are well known in the art.

As used herein, treatment means any manner in which the symptoms of acondition, disorder or disease are ameliorated or otherwise beneficiallyaltered. Hence treatment encompasses prophylaxis, therapy and/or cure.Treatment also encompasses any pharmaceutical use of the compositionsherein. Treatment also encompasses any pharmaceutical use of a modifiedFIX and compositions provided herein.

As used herein, amelioration of the symptoms of a particular disease ordisorder by a treatment, such as by administration of a pharmaceuticalcomposition or other therapeutic, refers to any lessening, whetherpermanent or temporary, lasting or transient, of the symptoms that canbe attributed to or associated with administration of the composition ortherapeutic.

As used herein, prevention or prophylaxis refers to methods in which therisk of developing disease or condition is reduced. Prophylaxis includesreduction in the risk of developing a disease or condition and/or aprevention of worsening of symptoms or progression of a disease orreduction in the risk of worsening of symptoms or progression of adisease.

As used herein an effective amount of a compound or composition fortreating a particular disease is an amount that is sufficient toameliorate, or in some manner reduce the symptoms associated with thedisease. Such amount can be administered as a single dosage or can beadministered according to a regimen, whereby it is effective. The amountcan cure the disease but, typically, is administered in order toameliorate the symptoms of the disease. Typically, repeatedadministration is required to achieve a desired amelioration ofsymptoms.

As used herein, “therapeutically effective amount” or “therapeuticallyeffective dose” refers to an agent, compound, material, or compositioncontaining a compound that is at least sufficient to produce atherapeutic effect. An effective amount is the quantity of a therapeuticagent necessary for preventing, curing, ameliorating, arresting orpartially arresting a symptom of a disease or disorder.

As used herein, “patient” or “subject” to be treated includes humans andor non-human animals, including mammals. Mammals include primates, suchas humans, chimpanzees, gorillas and monkeys; domesticated animals, suchas dogs, horses, cats, pigs, goats, cows; and rodents such as mice,rats, hamsters and gerbils.

As used herein, a combination refers to any association between two oramong more items. The association can be spatial or refer to the use ofthe two or more items for a common purpose.

As used herein, a composition refers to any mixture of two or moreproducts or compounds (e.g., agents, modulators, regulators, etc.). Itcan be a solution, a suspension, liquid, powder, a paste, aqueous ornon-aqueous formulations or any combination thereof.

As used herein, an “article of manufacture” is a product that is madeand sold. As used throughout this application, the term is intended toencompass modified protease polypeptides and nucleic acids contained inarticles of packaging.

As used herein, fluid refers to any composition that can flow. Fluidsthus encompass compositions that are in the form of semi-solids, pastes,solutions, aqueous mixtures, gels, lotions, creams and other suchcompositions.

As used herein, a “kit” refers to a packaged combination, optionallyincluding reagents and other products and/or components for practicingmethods using the elements of the combination. For example, kitscontaining a modified protease polypeptide or nucleic acid moleculeprovided herein and another item for a purpose including, but notlimited to, administration, diagnosis, and assessment of a biologicalactivity or property are provided. Kits optionally include instructionsfor use.

As used herein, antibody includes antibody fragments, such as Fabfragments, which are composed of a light chain and the variable regionof a heavy chain.

As used herein, a receptor refers to a molecule that has an affinity fora particular ligand. Receptors can be naturally-occurring or syntheticmolecules. Receptors also can be referred to in the art as anti-ligands.

As used herein, animal includes any animal, such as, but not limited to;primates including humans, gorillas and monkeys; rodents, such as miceand rats; fowl, such as chickens; ruminants, such as goats, cows, deer,sheep; ovine, such as pigs and other animals. Non-human animals excludehumans as the contemplated animal. The proteases provided herein arefrom any source, animal, plant, prokaryotic and fungal.

As used herein, gene therapy involves the transfer of heterologousnucleic acid, such as DNA, into certain cells, target cells, of amammal, particularly a human, with a disorder or condition for whichsuch therapy is sought. The nucleic acid, such as DNA, is introducedinto the selected target cells, such as directly or in a vector or otherdelivery vehicle, in a manner such that the heterologous nucleic acid,such as DNA, is expressed and a therapeutic product encoded thereby isproduced. Alternatively, the heterologous nucleic acid, such as DNA, canin some manner mediate expression of DNA that encodes the therapeuticproduct, or it can encode a product, such as a peptide or RNA that insome manner mediates, directly or indirectly, expression of atherapeutic product. Genetic therapy also can be used to deliver nucleicacid encoding a gene product that replaces a defective gene orsupplements a gene product produced by the mammal or the cell in whichit is introduced. The introduced nucleic acid can encode a therapeuticcompound, such as a protease or modified protease, that is not normallyproduced in the mammalian host or that is not produced intherapeutically effective amounts or at a therapeutically useful time.The heterologous nucleic acid, such as DNA, encoding the therapeuticproduct can be modified prior to introduction into the cells of theafflicted host in order to enhance or otherwise alter the product orexpression thereof. Genetic therapy also can involve delivery of aninhibitor or repressor or other modulator of gene expression.

As used herein, heterologous nucleic acid is nucleic acid that is notnormally produced in vivo by the cell in which it is expressed or thatis produced by the cell but is at a different locus or expresseddifferently or that mediates or encodes mediators that alter expressionof endogenous nucleic acid, such as DNA, by affecting transcription,translation, or other regulatable biochemical processes. Heterologousnucleic acid is generally not endogenous to the cell into which it isintroduced, but has been obtained from another cell or preparedsynthetically. Heterologous nucleic acid can be endogenous, but isnucleic acid that is expressed from a different locus or altered in itsexpression. Generally, although not necessarily, such nucleic acidencodes RNA and proteins that are not normally produced by the cell orin the same way in the cell in which it is expressed. Heterologousnucleic acid, such as DNA, also can be referred to as foreign nucleicacid, such as DNA. Thus, heterologous nucleic acid or foreign nucleicacid includes a nucleic acid molecule not present in the exactorientation or position as the counterpart nucleic acid molecule, suchas DNA, is found in a genome. It also can refer to a nucleic acidmolecule from another organism or species (i.e., exogenous).

Any nucleic acid, such as DNA, that one of skill in the art wouldrecognize or consider as heterologous or foreign to the cell in whichthe nucleic acid is expressed is herein encompassed by heterologousnucleic acid; heterologous nucleic acid includes exogenously addednucleic acid that also is expressed endogenously. Examples ofheterologous nucleic acid include, but are not limited to, nucleic acidthat encodes traceable marker proteins, such as a protein that confersdrug resistance, nucleic acid that encodes therapeutically effectivesubstances, such as anti-cancer agents, enzymes and hormones, andnucleic acid, such as DNA, that encodes other types of proteins, such asantibodies. Antibodies that are encoded by heterologous nucleic acid canbe secreted or expressed on the surface of the cell in which theheterologous nucleic acid has been introduced.

As used herein, a therapeutically effective product for gene therapy isa product that is encoded by heterologous nucleic acid, typically DNA,that, upon introduction of the nucleic acid into a host, a product isexpressed that ameliorates or eliminates the symptoms, manifestations ofan inherited or acquired disease or that cures the disease. Alsoincluded are biologically active nucleic acid molecules, such as RNAiand antisense RNA.

As used herein, recitation that a polypeptide “consists essentially” ofa recited sequence of amino acids means that only the recited portion,or a fragment thereof, of the full-length polypeptide is present. Thepolypeptide can optionally, and generally will, include additional aminoacids from another source or can be inserted into another polypeptide

As used herein, the singular forms “a,” “an” and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to compound, comprising “an extracellular domain”includes compounds with one or a plurality of extracellular domains.

As used herein, ranges and amounts can be expressed as “about” aparticular value or range. About also includes the exact amount. Hence“about 5 bases” means “about 5 bases” and also “5 bases.”

As used herein, “optional” or “optionally” means that the subsequentlydescribed event or circumstance does or does not occur, and that thedescription includes instances where said event or circumstance occursand instances where it does not. For example, an optionally substitutedgroup means that the group is unsubstituted or is substituted.

As used herein, the abbreviations for any protective groups, amino acidsand other compounds, are, unless indicated otherwise, in accord withtheir common usage, recognized abbreviations, or the IUPAC-IUBCommission on Biochemical Nomenclature (see, (1972) Biochem. 11:1726).

B. HEMOSTASIS AND ROLE OF FACTOR IX THEREIN

Provided herein are modified Factor IX (FIX) polypeptides, includingmodified FIXa polypeptides and catalytically active fragments thereof.Factor IX polypeptides play a role in the regulation of and process ofhemostasis, and hence can be used as therapeutic agents. Effectivedelivery of therapeutic proteins such as FIX for clinical use is a majorchallenge to pharmaceutical science. Once in the blood stream, theseproteins are constantly eliminated from circulation within a short timeby different physiological processes, involving metabolism as well asclearance using normal pathways for protein elimination, such as(glomerular) filtration in the kidneys or proteolysis in blood. Once inthe luminal gastrointestinal tract, these proteins are constantlydigested by luminal proteases. The latter can be a limiting processaffecting the half-life of proteins used as therapeutic agents inintravenous injection. Additionally, inhibitors in the blood canspecifically inhibit the activity of the therapeutic protein. Forexample, antithrombin (AT-III), heparin, and the AT-III/heparin complex,can inhibit the coagulant activity of FIX. More efficacious variants ofFIX with improved properties, including improved pharmacokinetic andpharmacodynamic properties, increased catalytic activity, and/orincreased resistance to inhibitors, are needed.

The modified FIX polypeptides provided herein exhibit improvedproperties, including improved pharmacokinetic properties, such asincreased serum half-life; increased resistance to inhibitors, such asantithrombin III (AT-III), heparin and the AT-III/heparin complex;increased catalytic activity; or any combination thereof. Hence,provided are modified FIX polypeptides that have increased coagulantactivity. Accordingly, these polypeptides have a variety of uses andapplications, for example, as therapeutics for modulating hemostasis.The following discussion provides a review of the coagulation processand the role of Factor IX in this process, before a discussion of FactorIX, and modifications thereof.

Hemostasis is the physiological mechanism that stems the bleeding thatresults from injury to the vasculature. Normal hemostasis depends oncellular components and soluble plasma proteins, and involves a seriesof signaling events that ultimately leads to the formation of a bloodclot. Coagulation is quickly initiated after an injury occurs to theblood vessel and endothelial cells are damaged. In the primary phase ofcoagulation, platelets are activated to form a haemostatic plug at thesite of injury. Secondary hemostasis follows involving plasmacoagulation factors, which act in a proteolytic cascade resulting in theformation of fibrin strands which strengthen the platelet plug.

Upon vessel injury, the blood flow to the immediate injured area isrestricted by vascular constriction allowing platelets to adhere to thenewly-exposed fibrillar collagen on the subendothelial connectivetissue. This adhesion is dependent upon the von Willebrand factor (vWF),which binds to the endothelium within three seconds of injury, therebyfacilitating platelet adhesion and aggregation. Activation of theaggregated platelets results in the secretion of a variety of factors,including ADP, ATP, thromboxane and serotonin. Adhesion molecules,fibrinogen, vWF, thrombospondin and fibronectin also are released. Suchsecretion promotes additional adhesion and aggregation of platelets,increased platelet activation and blood vessel constriction, andexposure of anionic phospholipids on the platelet surface that serve asplatforms for the assembly of blood coagulation enzyme complexes. Theplatelets change shape leading to pseudopodia formation, which furtherfacilitates aggregation to other platelets resulting in a loose plateletplug.

A clotting cascade of peptidases (the coagulation cascade) issimultaneously initiated. The coagulation cascade involves a series ofactivation events involving proteolytic cleavage. In such a cascade, aninactive protein of a serine protease (also called a zymogen) isconverted to an active protease by cleavage of one or more peptidebonds, which then serves as the activating protease for the next zymogenmolecule in the cascade, ultimately resulting in clot formation by thecross-linking of fibrin. For example, the cascade generates activatedmolecules such as thrombin (from cleavage of prothrombin), which furtheractivates platelets, and also generates fibrin from cleavage offibrinogen. Fibrin then forms a cross-linked polymer around the plateletplug to stabilize the clot. Upon repair of the injury, fibrin isdigested by the fibrinolytic system, the major components of which areplasminogen and tissue-type plasminogen activator (tPA). Both of theseproteins are incorporated into polymerizing fibrin, where they interactto generate plasmin, which, in turn, acts on fibrin to dissolve thepreformed clot. During clot formation, coagulation factor inhibitorsalso circulate through the blood to prevent clot formation beyond theinjury site.

The interaction of the system, from injury to clot formation andsubsequent fibrinolysis, is described below.

1. Platelet Adhesion and Aggregation

The clotting of blood is actively circumvented under normal conditions.The vascular endothelium supports vasodilation, inhibits plateletadhesion and activation, suppresses coagulation, enhances fibrincleavage and is anti-inflammatory in character. Vascular endothelialcells secrete molecules such as nitrous oxide (NO) and prostacylin,which inhibit platelet aggregation and dilate blood vessels. Release ofthese molecules activates soluble guanylate cyclases (sGC) andcGMP-dependent protein kinase I (cGKI) and increases cyclic guanosinemonophosphate (cGMP) levels, which cause relaxation of the smooth musclein the vessel wall. Furthermore, endothelial cells express cell-surfaceADPases, such as CD39, which control platelet activation and aggregationby converting ADP released from platelets into adenine nucleotideplatelet inhibitors. The endothelium also plays an important role in theregulation of the enzymes in the fibrinolytic cascade. Endothelial cellsdirectly promote the generation of plasmin through the expression ofreceptors of plasminogen (annexin II) and urokinase, as well as thesecretion of tissue-type and urokinase plasminogen activators, all ofwhich promote clot clearance. In a final layer of prothromboticregulation, endothelial cells play an active role in inhibiting thecoagulation cascade by producing heparan sulfate, which increases thekinetics of antithrombin III inhibition of thrombin and othercoagulation factors.

Under acute vascular trauma, however, vasoconstrictor mechanismspredominate and the endothelium becomes prothrombotic, procoagulatoryand proinflammatory in nature. This is achieved by a reduction ofendothelial dilating agents: adenosine, NO and prostacyclin; and thedirect action of ADP, serotonin and thromboxane on vascular smoothmuscle cells to elicit their contraction (Becker, Heindl et al. 2000).The chief trigger for the change in endothelial function that leads tothe formation of haemostatic thrombus is the loss of the endothelialcell barrier between blood and extracellular matrix (ECM) components(Ruggeri (2002) Nat Med 8:1227-1234). Circulating platelets identify anddiscriminate areas of endothelial lesions and adhere to the exposed subendothelium. Their interaction with the various thrombogenic substratesand locally-generated or released agonists results in plateletactivation. This process is described as possessing two stages, 1)adhesion: the initial tethering to a surface, and 2) aggregation: theplatelet-platelet cohesion (Savage et al. (2001) Curr Opin Hematol8:270-276).

Platelet adhesion is initiated when the circulating platelets bind toexposed collagen through interaction with collagen binding proteins onthe cell surface, and through interaction with vWF, also present on theendothelium. vWF protein is a multimeric structure of variable size,secreted in two directions by the endothelium; basolaterally and intothe bloodstream. vWF also binds to factor VIII, which is important inthe stabilization of factor VIII and its survival in the circulation.

Platelet adhesion and subsequent activation is achieved when vWF bindsvia its A1 domain to GPIb (part of the platelet glycoprotein receptorcomplex GPIb-IX-V). The interaction between vWF and GPIb is regulated byshear force such that an increase in the shear stress results in acorresponding increase in the affinity of vWF for GPIb. Integrin α1β2,also known on leukocytes as VLA-2, is the major collagen receptor onplatelets, and engagement through this receptor generates theintracellular signals that contribute to platelet activation. Bindingthrough α1β2 facilitates the engagement of the lower-affinity collagenreceptor, GP VI. This is part of the immunoglobulin superfamily and isthe receptor that generates the most potent intracellular signals forplatelet activation. Platelet activation results in the release ofadenosine diphosphate (ADP), which is converted to thromboxane A2.

Platelet activation also results in the surface expression of plateletglycoprotein IIb-Ma (GP IIb-IIIa) receptors, also known as plateletintegrin αIIbβ3. GP IIb-IIIa receptors allow the adherence of plateletsto each other (i.e. aggregation) by virtue of fibrinogen moleculeslinking the platelets through these receptors. This results in theformation of a platelet plug at the site of injury to help preventfurther blood loss, while the damaged vascular tissue releases factorsthat initiate the coagulation cascade and the formation of a stabilizingfibrin mesh around the platelet plug.

2. Coagulation Cascade

The coagulation pathway is a proteolytic pathway where each enzyme ispresent in the plasma as a zymogen, or inactive form. Cleavage of thezymogen is regulated to release the active form from the precursormolecule. The pathway functions as a series of positive and negativefeedback loops that control the activation process, where the ultimategoal is to produce thrombin, which can then convert soluble fibrinogeninto fibrin to form a clot. The coagulation factors, and other proteins,participate in blood coagulation through one or more of the intrinsic,extrinsic or common pathway of coagulation. As discussed below, thesepathways are interconnected, and blood coagulation likely occurs througha cell-based model of activation.

The generation of thrombin has historically been divided into threepathways, the intrinsic (suggesting that all components of the pathwayare intrinsic to plasma) and extrinsic (suggesting that one or morecomponents of the pathway are extrinsic to plasma) pathways that providealternative routes for the generation of activated factor X (FXa), andthe final common pathway which results in thrombin formation (FIG. 1).These pathways participate together in an interconnected andinterdependent process to effect coagulation. A cell-based model ofcoagulation was developed that describes these pathways (FIG. 2)(Hoffman et al. (2001) Thromb Haemost 85:958-965). In this model, the“extrinsic” and “intrinsic” pathways are effected on different cellsurfaces; the tissue factor (TF)-bearing cell and the platelet,respectively. The process of coagulation is separated into distinctphases, initiation, amplification and propagation, during which theextrinsic and intrinsic pathways function at various stages to producethe large burst of thrombin required to convert sufficient quantities offibrinogen to fibrin for clot formation.

a. Initiation

FVII is considered to be the coagulation factor responsible forinitiating the coagulation cascade, which initiation is dependent on itsinteraction with TF. TF is a transmembrane glycoprotein expressed by avariety of cells such as smooth muscle cells, fibroblasts, monocytes,lymphocytes, granulocytes, platelets and endothelial cells. Myeloidcells and endothelial cells only express TF when they are stimulated,such as by proinflammatory cytokines. Smooth muscle cells andfibroblasts, however, express TF constitutively. Accordingly, once thesecells come in contact with the bloodstream following tissue injury, thecoagulation cascade is rapidly initiated by the binding of TF withfactor VII or FVIIa in the plasma. TF/FVIIa complexes can be formed bythe direct binding of FVIIa to TF, or by the binding of FVII to TF andthen the subsequent activation of FVII to FVIIa by a plasma protease,such as FXa, FIXa, FXIIa, or FVIIa itself. The TF/FVIIa complex remainsanchored to the TF-bearing cell where it activates small amounts of FXinto FXa in what is known as the “extrinsic pathway” of coagulation.

The TF/FVIIa complex also cleaves small amounts of FIX into FIXa. FXaassociates with its cofactor FVa to also form a complex on theTF-bearing cell that can then covert prothrombin to thrombin. The smallamount of thrombin produced is, however, inadequate to support therequired fibrin formation for complete clotting. Additionally, anyactive FXa and FIXa are inhibited in the circulation by antithrombin III(AT-III) and other serpins, which are discussed in more detail below.This would normally prevent clot formation in the circulation. In thepresence of injury, however, damage to the vasculature results inplatelet aggregation and activation at this site of thrombin formation,thereby allowing for amplification of the coagulation signal.

b. Amplification

Amplification takes place when thrombin binds to and activates theplatelets. The activated platelets release FV from their alpha granules,which is activated by thrombin to FVa. Thrombin also releases andactivates FVIII from the FVIII/vWF complex on the platelet membrane, andcleaves FXI into FXIa. These reactions generate activated platelets thathave FVa, FVIIIa and FIXa on their surface, which set the stage for alarge burst of thrombin generation during the propagation stage.

c. Propagation

Propagation of coagulation occurs on the surface of large numbers ofplatelets at the site of injury. As described above, the activatedplatelets have FXIa, FVIIIa and FVa on their surface. It is here thatthe extrinsic pathway is effected. FXIa activates FIX to FIXa, which canthen bind with FVIIIa. This process, in addition to the small amount ofFIXa that is generated by cleavage of FIX by the TF/FVIIa complex on theTF-bearing cell, generates a large amount FIXa in complex with itscofactor, FVIIIa, calcium and a suitable phospholipid surface. Thiscomplex is termed the tenase or Xase complex, and it cleaves andactivates the Factor X (FX) to Factor Xa (FXa). The FXa molecules bindto FVa to generate the prothrombinase complexes that activateprothrombin to thrombin. Thrombin acts in a positive feedback loop toactivate even more platelets and again initiates the processes describedfor the amplification phase.

Very shortly, there are sufficient numbers of activated platelets withthe appropriate complexes to generate the burst of thrombin that islarge enough to generate sufficient amounts of fibrin from fibrinogen toform a hemostatic fibrin clot. Fibrinogen is a dimer soluble in plasmawhich, when cleaved by thrombin, releases fibrinopeptide A andfibrinopeptide B. Fibrinopeptide B is then cleaved by thrombin, and thefibrin monomers formed by this second proteolytic cleavage spontaneouslyforms an insoluble gel. The polymerized fibrin is held together bynoncovalent and electrostatic forces and is stabilized by thetransamidating enzyme factor XIIIa (FXIIIa), produced by the cleavage ofFXIII by thrombin. Thrombin also activates TAFI, which inhibitsfibrinolysis by reducing plasmin generation at the clot surface.Additionally, thrombin itself is incorporated into the structure of theclot for further stabilization. These insoluble fibrin aggregates(clots), together with aggregated platelets (thrombi), block the damagedblood vessel and prevent further bleeding.

3. Regulation of Coagulation

During coagulation, the cascade is regulated by constitutive andstimulated processes to inhibit further clot formation. Regulation isimportant to a) limit ischemia of tissues by fibrin clot formation, andb) prevent widespread thrombosis by localizing the clot formation onlyto the site of tissue injury.

Regulation is achieved by the actions of several inhibitory molecules.For example, antithrombin III (AT-III) and tissue factor pathwayinhibitor (TFPI) work constitutively to inhibit factors in thecoagulation cascade. TFPI predominantly inhibits FXa and FVIIa/TFcomplex. In contrast, AT-III, which is a serine protease inhibitor(serpin), predominantly inhibits thrombin, FXa, and FIXa. The inhibitionof these coagulation factors by AT-III is enhanced greatly by heparin,which binds AT-III to induce an activating conformational change thataccelerates the inhibitory reaction. Heparin also can inhibit theactivity of the FIXa/FVIIIa complex in an AT-III-independent manner(Yuan et al., (2005) Biochemistry 44:3615-3625). An additional factor,Protein C, which is stimulated via platelet activation, regulatescoagulation by proteolytic cleavage and inactivation of FVa and FVIIIa.Protein S enhances the activity of Protein C. Further, another factorwhich contributes to coagulation inhibition is the integral membraneprotein thrombomodulin, which is produced by vascular endothelial cellsand serves as a receptor for thrombin. Binding of thrombin tothrombomodulin inhibits thrombin procoagulant activities and alsocontributes to protein C activation.

Fibrinolysis, the breakdown of the fibrin clot, also provides amechanism for regulating coagulation. The crosslinked fibrin multimersin a clot are broken down to soluble polypeptides by plasmin, a serineprotease. Plasmin can be generated from its inactive precursorplasminogen and recruited to the site of a fibrin clot in two ways: byinteraction with tissue plasminogen activator (tPA) at the surface of afibrin clot, and by interaction with urokinase plasminogen activator(uPA) at a cell surface. The first mechanism appears to be the major oneresponsible for the dissolution of clots within blood vessels. Thesecond, although capable of mediating clot dissolution, can play a majorrole in tissue remodeling, cell migration, and inflammation.

Clot dissolution also is regulated in two ways. First, efficient plasminactivation and fibrinolysis occur only in complexes formed at the clotsurface or on a cell membrane, while proteins free in the blood areinefficient catalysts and are rapidly inactivated. Second, plasminogenactivators and plasmin are inactivated by molecules such as plasminogenactivator inhibitor type 1 (PAI-1) and PAI-2 which act on theplasminogen activators, and α2-antiplasmin and α 2-macroglobulin thatinactivate plasmin. Under normal circumstances, the timely balancebetween coagulation and fibrinolysis results in the efficient formationand clearing of clots following vascular injury, while simultaneouslypreventing unwanted thrombotic or bleeding episodes.

C. FACTOR IX (FIX) STRUCTURE AND FUNCTION

Provided herein are modified FIX polypeptides with improved activitiesor functions. FIX is a polypeptide that is involved in the coagulationcascade. The role of FIX in the coagulation cascade is related to itsstructure and mechanism of activation. It is understood that themodulation of coagulation by modified FIX polypeptides provided hereinalso is linked to its structure and mechanism of activation. Thesefeatures can be the same as an unmodified FIX polypeptide. In othercases, these features can be modified in a FIX polypeptide providedherein, thus resulting in a polypeptide with altered or improvedactivities or properties. For example, modification of a FIX polypeptidecan alter one or more activities of a FIX polypeptide. For example,provided are modified FIX polypeptides that exhibit increased levels ofglycosylation compared to a wild-type FIX polypeptide. The modified FIXpolypeptides can thus exhibit improved pharmacokinetic properties, suchas reduced clearance and increased serum half-life compared to awild-type FIX polypeptide, when tested using in vivo assays. Alsoprovided are modified FIX polypeptides that exhibit increased resistanceto inhibitors, such as AT-III, heparin and the AT-III/heparin complex;and/or increased catalytic activity. Thus, provided are modified FIXpolypeptides that exhibit improved therapeutic properties compared to anunmodified FIX polypeptide. A summary of structural and functionalfeatures of FIX polypeptides and modified FIX polypeptides are describedbelow.

Factor IX is a vitamin K-dependent serine protease and is an importantcoagulation factor in hemostasis. It is synthesized as a single chainzymogen in the liver and circulates in the blood in this inactivatedstate until activated as part of the coagulation cascade. Followingactivation from the FIX zymogen to activated FIX (FIXa) by FXIa or theTF/FVIIa complex, FIXa binds its cofactor, FVIIIa. The resultingFIXa/FVIIIa complex binds and activates FX to FXa, thus continuing thecoagulation cascade described above to establish hemostasis. Theconcentration of FIX in the blood is approximately 4-5 μg/mL, and it hasa half-life of approximately 18-24 hours.

Hemophilia B, also known as Christmas disease or factor IX deficiency,is caused by a deficiency or dysfunction of FIX resulting from any oneor more of a variety of mutations in the FIX gene. While less prevalentthan Hemophilia A, Hemophilia B remains a significant disease in whichrecurrent joint bleeds can lead to synovial hypertrophy, chronicsynovitis, with destruction of synovium, cartilage, and bone leading tochronic pain, stiffness of the joints, and limitation of movementbecause of progressive severe joint damage. Recurrent muscle bleeds alsoproduce acute pain, swelling, and limitation of movement, while bleedingat other sites can contribute to morbidity and mortality. Treatment istypically by replacement therapy with recombinant FIX (rFIX). Providedherein are modified FIX polypeptides that are designed to have increasedcoagulation activity upon activation, and that can serve as improvedtherapeutics to treat diseases and conditions amenable to factor IXtherapy, such as Hemophilia B.

1. FIX Structure

The human FIX gene is located on the X chromosome and is approximately34 kb long with eight exons. The human FIX transcript is 2803nucleotides and contains a short 5′ untranslated region, an open readingframe (including stop codon) of 1383 nucleotides, and a 3′ untranslatedregion. The 1383 nucleotide open reading frame (or FIX mRNA; SEQ IDNO:1) encodes a 461 amino acid precursor polypeptide (Swiss-Protaccession no. P00740; SEQ ID NO:2) containing a 28 amino acid N-terminalsignal peptide (amino acids 1-28 of SEQ ID NO:2) that directs the factorIX polypeptide to the cellular secretory pathway. In addition thehydrophobic signal peptide, the FIX precursor polypeptide also containsan 18 amino acid propeptide (aa 29-46 of SEQ ID NO:2) that, whencleaved, releases the 415 amino acid mature polypeptide (SEQ ID NO:3)that circulates in the blood as a zymogen until activation to FIXa. Inaddition to the signal peptide and propeptide, the FIX precursor alsocontains the following segments and domains: a Gla domain (aa 47-92 ofSEQ ID NO:2, corresponding to aa 1-46 of the mature FIX protein setforth in SEQ ID NO:3), epidermal growth factor (EGF)-like domain 1(EGF1; aa 93-129 of SEQ ID NO:2, corresponding to aa 47-83 of the matureFIX protein set forth in SEQ ID NO:3), EGF2 (aa 130-171 of SEQ ID NO:2,corresponding to aa 84-125 of the mature FIX protein set forth in SEQ IDNO:3), a light chain (aa 47-191 of SEQ ID NO:2, corresponding to aa1-145 of the mature FIX protein set forth in SEQ ID NO:3), an activationpeptide (aa 192-226 of SEQ ID NO:2, corresponding to aa 146-180 of themature FIX protein set forth in SEQ ID NO:3), a heavy chain (aa 227-461of SEQ ID NO:2, corresponding to aa 181-415 of the mature FIX proteinset forth in SEQ ID NO:3) and a serine protease domain (aa 227-459 ofSEQ ID NO:2, corresponding to aa 181-413 of the mature FIX protein setforth in SEQ ID NO:3).

Like other vitamin K-dependent proteins, such as prothrombin,coagulation factors VII and X, and proteins C, S, and Z, the Gla domainof FIX is a membrane binding motif which, in the presence of calciumions, interacts with the phospholipid membranes of cells. The vitaminK-dependent proteins require vitamin K for the posttranslationalsynthesis of γ-carboxyglutamic acid, an amino acid clustered in the Gladomain of these proteins. The FIX Gla domain has 12 glutamic acidresidues, each of which are potential carboxylation sites. Many of themare, therefore, modified by carboxylation to generate γ-carboxyglutamicacid residues. There are a total of eight Ca²⁺ binding sites, of bothhigh and low affinity, in the FIX Gla domain that, when occupied bycalcium ions, facilitate correct folding of the Gla domain to exposehydrophobic residues in the FIX polypeptide that are inserted into thelipid bilayer to effect binding to the membrane.

In addition to the Gla domain, the FIX polypeptide also contains twoEGF-like domains. Each EGF-like domain contains six highly conservedcysteine residues that form three disulphide bonds in each domain in thesame pattern observed in the EGF protein. The first EGF-like domain(EGF1) is a calcium-binding EGF domain containing a high affinity Ca²⁺binding site (Rao et al., (1995) Cell 82:131-141) that, when occupied bya calcium ion, contributes to the correct folding of the molecule andpromotes biological activity. The second EGF domain (EGF2) does notcontain a calcium binding site.

The serine protease domain, or catalytic domain, of FIX is the domainresponsible for the proteolytic activity of FIXa. Like other serineproteases, FIX contains a serine protease catalytic triad composed ofH221, D269 and S365 (corresponding to H57, D102 and S195 by chymotrypsinnumbering).

Activation of mature FIX to FIXa is effected by proteolytic cleavage ofthe R145-A146 bonds and R180-V181 bonds (numbering relative to themature FIX polypeptide set forth in SEQ ID NO:3), releasing theactivation peptide that corresponds to aa 146-180 of the mature FIXprotein set forth in SEQ ID NO:3. Thus, following activation, FIXaconsists of two chains; the light chain and heavy chain. The light chaincontains the Gla domain, EGF1 and EGF2 domains, and the heavy chaincontains the protease domain. The two chains are held together by asingle disulphide bond between C132 and C289.

2. FIX Post-Translational Modification

The Factor IX precursor polypeptide undergoes extensiveposttranslational modification to become the mature zymogen that issecreted into the blood. Such posttranslation modifications includeγ-carboxylation, β-hydroxylation, cleavage of the signal peptide andpropeptide, O- and N-linked glycosylation, sulfation andphosphorylation. The N-terminal signal peptide directs the polypeptideto the endoplasmic reticulum (ER), after which it is cleaved.Immediately prior to secretion from the cell, the propeptide is cleavedby processing proteases, such as, for example, PACE/furin, thatrecognize at least two arginine residues within four amino acids priorto the cleavage site.

A single enzyme, vitamin K-dependent gamma-carboxylase, catalyzes theγ-carboxylation FIX in the ER (Berkner (2000) J. Nutr. 130:1877-80). Inthe carboxylation reaction, the γ-carboxylase binds to the FIXpropeptide and catalyzes a second carboxylation on the γ-carbon of theglutamic acid residues (i.e. Glu to γ-carboxyglutamyl or Gla) in the Gladomain of the polypeptide. Assuming all glutamic acid residues areγ-carboxylated, FIX contains 12 Gla residues, where the first 10 are athomologous positions of other vitamin K-dependent proteins. The Gladomain of FIX then processively carboxylates all glutamates in thecluster before releasing the substrate (Morris et al. 1995; Berkner2000; Stenina et al. 2001).

FIX also is partially β-hydroxylated. This modification is performed bya dioxygenase, which hydroxylates the β-carbon of D64 (corresponding tothe mature FIX polypeptide set forth in SEQ ID NO:3) in EGF1.Approximately one third of human FIX polypeptides are β-hydroxylated.Although D64 contributes to the high affinity Ca²⁺ binding site in theEGF1 domain of FIX, the hydroxylation of this residue does not appear tobe necessary for Ca²⁺ binding, nor for biological activity (Derian etal., (1989) J. Biol. Chem. 264:6615-6618, Sunnerhagen et al., (1993) J.Biol. Chem. 268: 23339-23344). Additional post-translationalmodifications include sulfonation at the tyrosine at position 155, andphosphorylation at the serine residue at position 158. Thesepost-translational modifications of Factor IX have been implicated incontributing to in vivo recovery of FIX (Kaufman (1998) Thromb. Haemost.79:1068-1079, U.S. Pat. No. 7,575,897).

FIX is N-linked glycosylated at asparagine residues in the activationpeptide corresponding to N157 and N167 of the mature FIX polypeptide setforth in SEQ ID NO:3. Post-translational modification also results inthe serine residue at position 53 (corresponding to the mature FIXpolypeptide set forth in SEQ ID NO:3) having O-linked disaccharides andtrisaccharides, while the serine residue at position 61 contains anO-linked tertrasaccharide. (Nishimura et al., (1989) J Biol. Chem.264:20320-20325, Harris et al., (1993) Biochemistry 32:6539-6547).Additionally, the threonine residues at amino acid positions 159 and 169(corresponding to the mature FIX polypeptide set forth in SEQ ID NO:3)are O-glycosylated (Agarwala et al., (1994) Biochemistry 33:5167-5171).The threonine residues at amino acid positions 172 and 179 also may beO-glycosylated.

3. FIX Activation

Factor IX circulates predominantly as a zymogen with minimal proteolyticactivity until it is activated by proteolytic cleavage. Activation canbe effected by the TF/FVIIa complex or Factor XIa. Activation byTF/FVIIa is through the intrinsic pathway, while activation by FXIa isthrough the extrinsic pathway, described above. The process ofactivation appears to be sequential with initial cleavage of theArg145-Ala146 bond, followed by cleavage of the Arg180-Val181 bond(Schmidt et al. (2003) Trends Cardio. Med. 13:39-45). The proteolyticcleavage releases the activation peptide, forming the two-chain FIXamolecule containing the light chain (corresponding to amino acidpositions 1-145 of SEQ ID NO:3) and heavy chain (corresponding to aminoacid positions 181-415 of SEQ ID NO:3) held together by a disulphidebond between the two cysteines at amino acid positions 132 and 289(numbering corresponding to the mature FIX polypeptide set forth in SEQID NO:3).

At least two exosites in FX appear to be involved in binding to TF inthe TF/FVIIa complex to form the FIX/TF/FVIIa ternary complex (Chen etal., (2002) Thromb. Haemost. 88:74-82). Studies suggest that the EGF1domain of FIX is required for FIX activation by the TF/FVIIa complex.For example, mutation of G48 (relative to the mature FIX polypeptide setforth in SEQ ID NO:3) in the EGF1 domain of FIX reduces its activationby TF/FVIIa (Wu et al., (2000) Thromb. Haemost. 84:626-634). Further,the EGF1 domain of FIX has been shown to interact with TF in theTF/FVIIa complex (Zhong et al., (2002) J. Biol. Chem 277:3622). Incontrast, however, the EGF1 domain does not appear to be required forFIX activation by FXIa. The Gla domain also is involved in binding tothe TF/FVIIa complex and, therefore, in activation. The Gla domain ofFIX interacts with the same region in TF as FX, which also is activatedby the TF/FVIIa complex (Kirchhofer et al., (2000) Biochem.39:7380-7387).

Following cleavage and release of the activation peptide, a new aminoterminus at V181 (corresponding to the mature FIX polypeptide set forthin SEQ ID NO:3; V16 by chymotrypsin numbering) is generated. Release ofthe activation peptide facilitates a conformational change whereby theamino group of V181 inserts into the active site and forms a salt bridgewith the side chain carboxylate of D364. Such a change is required forconversion of the zymogen state to an active state, as the changeconverts the hydroxyl side chain of S365 to a reactive species that isable to hydrolyze the cleavage site of its substrate, FX. The activatedFIXa polypeptide remains in a zymogen-like conformation until additionalconformational changes are induced, such as by binding with FVIIIa, togenerate a FIXa polypeptide with maximal catalytic activity.

4. FIX Function

FIX plays an important role in the coagulation pathway and a deficiencyor absence of FIX activity leads to hemophilia B. Once activated fromFIX to FIXa, FIXa in turn functions to activate the large amounts of FXto FXa that are required for coagulation. To do so, FIXa must first bindto its cofactor, Factor VIIIa, to form the FIXa/FVIIIa complex, alsocalled the intrinsic tenase complex, on the phospholipid surface of theactivated platelet. Both the Gla domain and EGF2 domain of FIX areimportant for stable binding to phospholipids. The FIXa/FVIIIa complexthen binds FX to cleave this coagulation factor to form FIXa.

FIXa is virtually inactive in the absence of its cofactor, FVIIIa, andphysiologic substrate, FX. Experimental studies indicate that this canbe attributed mainly to the 99-loop. When FIXa is not bound by itscofactor, Y177 locks the 99-loop in an inactive conformation in whichthe side chains of Y99 and K98 (by chymotrypsin numbering, correspondingto Y266 and K265 of the mature FIX polypeptide set forth in SEQ ID NO:3)impede substrate binding. Binding of FVIIIa to FIXa unlocks and releasesthis zymogen-like conformation, and FX is then able to associate withthe FIXa/FVIIIa complex and rearrange the unlocked 99-loop, subsequentlybinding to the active site cleft (Sichler et al., (2003) J. Biol. Chem.278:4121-4126). The binding of FIXa to phospholipids and the presence ofCa²⁺ further enhances the reaction.

Several models of the FIXa/FVIIIa interaction have been proposed (seee.g. Autin et al., (2005) J. Thromb. Haemost. 3:2044-2056,Stoilova-McPhie et al., (2002) Blood 99: 1215-1223, Bajaj et al., (2001)J. Biol. Chem. 276:16302-16309, Schmidt et al., (2003) TrendsCardiovasc. Med. 13:39-45). FIXa binds to FVIIIa in an interactioninvolving more than one domain of the FIXa polypeptide. FVIIIa is aheterodimer composed of three noncovalently associated chains: A1, A2and A3-C1-C2. A3-C1-C2 also is referred to as the light chain. Theprotease domain of FIXa appears to interact with the A2 subunit ofFVIIIa. Studies suggest that the 293-helix (126-helix by chymotrypsinnumbering), 330-helix (162-helix by chyotrypsin numbering) and N346(N178) by chymotrypsin numbering) of FIXa are involved in theinteraction with the A2 subunit of FVIIIa. The EGF1/EGF2 domains of FIXainteract with the A3 subunit of FVIIIa. Further, it is postulated thatthe Gla domain of FIXa interacts with the C2 domain of FVIIIa. Calciumions and phospholipids also contribute to binding of FIXa and FVIIIa.For example, the presence of phospholipids increases the binding of FIXato FVIIIa by approximately 2000-fold (Mathur et al., (1997) J. Biol.Chem. 272:). Following binding of FX by the FIXa/FVIIIa complex, theprotease domain (or catalytic domain) of FIXa is responsible forcleavage of FX at R194-1195 to form FXa.

The activity of FIXa is regulated by inhibitory molecules, such as theAT-III/heparin complex, as discussed above, and other clearancemechanisms, such as the low-density lipoprotein receptor-related protein(LRP). LRP is a membrane glycoprotein that is expressed on a variety oftissues, including liver, brain, placenta and lung. LRP binds a widerange of proteins and complexes in addition to FIXa, including, but notlimited to, apolipoproteins, lipases, proteinases, proteinase-inhibitorcomplexes, and matrix proteins. The zymogen or inactive form of FIX doesnot bind LRP. Rather, upon activation, an LRP-binding site is exposed(Neels et al., (2000) Blood 96:3459-3465). This binding site is locatedin a loop in the protease domain spanning residues 342 to 346 of themature FIX polypeptide set forth in SEQ ID NO:3 (Rohlena et al., (2003)J. Biol. Chem. 278:9394-9401).

5. FIX as a Biopharmaceutical

Factor IX is integrally involved in the blood coagulation process,where, in its activated form (FIXa), it forms a tenase complex withFVIIIa and activates FX to FXa. FXa, in conjunction with phospholipids,calcium and FVa, converts prothrombin to thrombin, which in turn cleavesfibrinogen to fibrin monomers, thus facilitating the formation of arigid mesh clot. Many studies have demonstrated the ability of exogenousFIX to promote blood clotting in patients with hemophilia. For example,hemophilia B patients, who are deficient in FIX, can be treated byreplacement therapy with exogenous FIX. Early replacement therapiesutilized plasma purified FIX, such as therapeutics marketed as MonoNine®Factor IX and Alpha-nine-SD® Factor IX. Plasma purified FIX complextherapeutics also have been used, including Bebulin® VH, a purifiedconcentrate of FIX with FX and low amounts of FVII; Konyne® 80 (Bayer),a purified concentrate of FIX, with FII, FX, and low levels of FVII;PROPLEX® T (Baxter International), a heat treated product prepared frompooled normal human plasma containing FIX with FII, FVII, and FX; andProfilnine SD® (Alpha Therapeutic Corporation). More recently, however,a human recombinant Factor IX (BeneFIX® Coagulation Factor IX(Recombinant), Wyeth) has been approved for use in the control andprevention of bleeding episodes in hemophilia B patients, includingcontrol and prevention of bleeding in surgical settings. BeneFIX®Coagulation Factor IX (Recombinant) has an amino acid sequence set forthin SEQ ID NO:20, and is identical to the Ala148 allelic form ofplasma-derived Factor IX. Thus, compared to the wild-type FIXpolypeptide set forth in SEQ ID NO:3, BeneFIX®, Coagulation Factor IX(Recombinant) contains a T148A mutation.

In addition to its use as a procoagulant, inactive forms of FIX, orforms with reduced catalytic activity, can be used as an anticoagulant,such as in the treatment of thrombotic diseases and conditions.

Typically, FIX is administered intravenously, but also can beadministered orally, systemically, buccally, transdermally,intramuscularly and subcutaneously. FIX can be administered once ormultiple times. Generally, multiple administrations are used intreatment regimens with FIX to effect coagulation.

As discussed herein below, modified FIX polypeptides provided hereinalso can be used in any treatment or pharmaceutical method in which anunmodified or wildtype or other therapeutically active FIX polypeptideis known to be used. In such uses, methods and processes, the modifiedFIX polypeptides provided herein exhibit improved properties compared toa wildtype or the unmodified FIX polypeptide.

D. MODIFIED FIX POLYPEPTIDES

Provided herein are modified factor IX polypeptides. The FIXpolypeptides can be modified by deletions, insertions or replacement(substitution) of one or more amino acid residues in the primarysequence of a wildtype or unmodified FIX polypeptide. The resultingmodified polypeptides exhibit improved properties or activities comparedto the unmodified or wildtype FIX polypeptide. For example, the modifiedfactor IX polypeptides, including modified FIXa polypeptides andfragments of modified factor IX and factor IXa polypeptides, can havealtered posttranslational modification, such as altered glycosylation,including hyperglycosylation, and/or altered phosphorylation orsulfation, such as decreased phosphorylation or sulfation; increasedresistance to inhibitors, such as AT-III and/or heparin; decreasedbinding to LRP; increased catalytic activity; improved pharmacokineticproperties, including decreased clearance and increased serum half-lifein vivo; increased coagulant activity; or any combination thereof.Typically, the modified FIX polypeptides exhibit procoagulant activity.Thus, provided herein are modified FIX polypeptides that exhibitincreased coagulant activity upon activation from their single-chainzymogen form and subsequent binding to the cofactor, FVIIIa. Suchmodified FIX polypeptides can be administered to patients with diseasesor conditions characterized by insufficient coagulation, such as, forexample, hemophilia B.

In some examples, the modified FIX polypeptides provided herein exhibitincreased resistance to inhibitors, including AT-III, heparin and theAT-III/heparin complex, compared to an unmodified FIX polypeptide. Suchmodified FIX polypeptides can exhibit increased coagulant activitycompared to an unmodified FIX polypeptide. In further examples, themodified factor IX polypeptides provided herein exhibit alteredposttranslation modification, such as altered glycosylation levelsand/or altered types of glycosylation compared to an unmodified FIXpolypeptide.

In some examples, the modified FIX polypeptides provided herein exhibitincreased glycosylation compared to an unmodified FIX polypeptide. Thus,provided herein are hyperglycosylated FIX polypeptides. The modified FIXpolypeptides can exhibit increased glycosylation by virtue of theincorporation of at least one non-native glycosylation site (i.e. aglycosylation site that is not found in the unmodified or wild-type FIXpolypeptide) to which a carbohydrate moiety is linked. Such modified FIXpolypeptides can exhibit improved pharmacokinetic properties in vivo,including decreased clearance and increased serum half-life. Theintroduction of a non-native glycosylation site and subsequentcarbohydrate moiety can further improve the activity of the modified FIXpolypeptide by sterically hindering the interaction of the FIXpolypeptide with one or more other proteins. For example, aglycosylation site can be introduced such that when a carbohydratemoiety is attached at this site, it sterically hinders the interactionof the modified FIX polypeptide with the AT-III/heparin complex,resulting in a polypeptide with increased resistance to AT-III/heparin.This can further reduce clearance of the polypeptide from thecirculation. Thus, the effects of the introduction of a newglycosylation site can be several-fold if the carbohydrate moiety alsosterically hinders an interaction with another protein(s), such as theAT-III/heparin complex.

For example, the modified FIX polypeptides provided herein can containone or more modifications that introduce one or more non-nativeglycosylation sites compared to the unmodified FIX polypeptide. Forexample, 1, 2, 3, 4, 5, 6, or more non-native glycosylation sites can beintroduced. Glycosylation sites that can be introduced include, but arenot limited to, N-glycosylation sites, O-glycosylation sites, or acombination thereof. Thus, when produced in a cell that facilitatesglycosylation, or following in vitro glycosylation, the modified FIXpolypeptides provided herein can contain 1, 2, 3, 4, 5, 6 or morecarbohydrate moieties, each linked to different non-native glycosylationsites, in addition to the carbohydrate moieties linked to the nativeglycosylation sites (e.g. the native glycosylation sites correspondingto S53, S61, N157, N167, T159, T169, T172 and T179 of the mature FIXpolypeptide set forth in SEQ ID NO:3). In a particular example, themodified FIX polypeptides provided herein contain one or more non-nativeN-glycosylation sites. Thus, the modified FIX polypeptides can exhibitincreased levels of N-glycosylation compared to an unmodified FIXpolypeptide.

The modified FIX polypeptides with increased glycosylation also canexhibit, for example, increased solubility, increased AT-III/heparinresistance, increased serum half-life, decreased immunogenicity and/orincreased coagulant activity compared to an unmodified FIX polypeptide.Such modified FIX polypeptides can be used in the treatment of bleedingdisorders or events, such as hemophilias or injury, where the FIXpolypeptides can function to promote blood coagulation. In someinstances, the modified FIX polypeptides provided herein that exhibitincreased glycosylation also can contain one or more modifications thatrender the protein inactive, or mostly inactive. Such polypeptides,therefore, can exhibit increased anti-coagulant activity and can be usedin the treatment of thrombotic events, conditions or diseases.Typically, however, the modified FIX polypeptides provided herein areprocoagulants.

The modified FIX polypeptides provided herein also can exhibit otheractivities and/or properties. For example, some of the modified FIXpolypeptides contain one or more modifications that increase catalyticactivity. In other examples, the modified FIX polypeptides contain oneor more modifications that decrease phosphorylation, sulfation,hydroxylation and/or glycosylation. In further examples, the modifiedFIX polypeptides contain modifications that interfere with theinteraction between FIX and LRP. By interrupting the binding of FIX toLRP, the clearance of FIX from circulation can be decreased. Hence,modifications that reduce the binding of FIX to LRP can improve thepharmacokinetic properties of FIX in vivo.

The modifications, such as amino acid replacements, described herein,such as those modifications that introduce one or more non-nativeglycosylation sites or increase resistance to inhibitors, can be made inany FIX polypeptide (e.g. unmodified or wildtype FIX polypeptide),including a precursor FIX polypeptide with a sequence set forth in SEQID NO:2, a mature FIX polypeptide set forth in SEQ ID NO:3, or in a FIXpolypeptide having a sequence of amino acids that exhibits at least 40%,50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity to the FIX polypeptide set forth in SEQ ID NOS:2 orSEQ ID NO:3. It is understood that reference herein to amino acidresidues is with respect to the numbering of the mature FIX polypeptideset forth in SEQ ID NO:3. It is within the level of one of skill in theart to identify a corresponding amino acid residue in another FIXpolypeptide of any form, such as a precursor, mature or other activeform, by alignment of the sequence of the other FIX polypeptide with SEQID NO:3 (see e.g. FIGS. 3A-3D). Any amino acid replacement providedherein can be made at a corresponding amino acid residue that differs oris not the same as the replacement amino acid residue. It is within thelevel of one of skill in the art to test any resulting modified FIXpolypeptide for activity or property as described herein.

For example, the modifications, such as an amino acid replacement, canbe made in any species, allelic or modified variant, such as thosedescribed in the art. Allelic variants of FIX include, but are notlimited to, T148A and T412P. Any of the amino acid replacements providedherein can be a Factor IX that contains mutations T148A or T412P. Forexample, the modifications such as any amino acid replacement, can bemade in a FIX polypeptide set forth in SEQ ID NO:325 or SEQ ID NO:20.Exemplary species variants for modification herein include, but are notlimited to, human and non-human polypeptides including FIX polypeptidesfrom chimpanzee, rhesus macaque, mouse, rat, guinea pig, pig, dog, cat,rabbit, chicken, cow, sheep, frog, zebrafish and Japanese pufferfish FIXpolypeptides, whose sequences are set forth in SEQ ID NOS:4-18,respectively. Modifications in a FIX polypeptide can be made to a FIXpolypeptide that also contains other modifications, such as thosedescribed in the art, including modifications of the primary sequenceand modifications not in the primary sequence of the polypeptide (seee.g. Section D.6, which describes exemplary modified FIX polypeptides towhich the amino acid modifications described herein can be made).

In other examples, the modifications, such as an amino acid replacement,can be made in any active fragment of a FIX polypeptide, such as anactive fragment of SEQ ID NO:2 or SEQ ID NO:3, or an active fragment ofa species, allelic or modified variant, such as those described in theart. The active fragment contains a contiguous sequence of amino acidscontaining the catalytically active domain of the polypeptide or acatalytically active portion thereof containing the amino acidmodifications, such as amino acid replacements describes herein. Theactive fragment exhibit at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%or more of the activity of the mature form of the polypeptide, such asthe FIX polypeptide set forth in SEQ ID NO:3.

Modification of FIX polypeptides also include modification ofpolypeptides that are hybrids of different FIX polypeptides and alsosynthetic FIX polypeptides prepared recombinantly or synthesized orconstructed by other methods known in the art based upon the sequence ofknown polypeptides. For example, based on alignment of FIX with othercoagulation factor family members, including, but not limited to, factorFVII (FVII) and factor X (FX), homologous domains among the familymembers are readily identified. Chimeric variants of FIX polypeptidescan be constructed where one or more amino acids or entire domains arereplaced in the FIX amino acid sequence using the amino acid sequence ofthe corresponding family member. Additionally, chimeric FIX polypeptidesinclude those where one or more amino acids or entire domains arereplaced in the human FIX amino acid sequence using the amino acidsequence of a different species. Such chimeric proteins can be used asthe starting, unmodified FIX polypeptide herein.

Modifications provided herein of a starting, unmodified referencepolypeptide include amino acid replacements or substitutions, additionsor deletions of amino acids, or any combination thereof. For example,modified FIX polypeptides include those with 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50 or more modifiedpositions. In some examples, a modification that is made to alter oneactivity or property of FIX also can, or instead, affect one more otheractivities or properties. For example, a modification made to increaseresistance to inhibitors also, or instead, can increase catalyticactivity. In another example, a modification made to introduce a newglycosylation site also can result in increased resistance to inhibitorsand/or increased catalytic activity. In a further example, amodification made to decrease binding to LRP can also, or instead,increase resistance to an inhibitor, such as AT-III/heparin. Thus,although the modifications described herein typically are described inrelation to their intended affect on FIX activities or properties, it isunderstood that any of the modifications described herein, alone or inconjunction with one or more other modifications, can result in changesin other, unpredicted, activities or properties.

Any modification provided herein can be combined with any othermodification known to one of skill in the art. Typically, the resultingmodified FIX polypeptide exhibits increased coagulation activity when itis in its two-chain form. The activities or properties that can bealtered as a result of modification include, but are not limited to,coagulation or coagulant activity; pro-coagulant activity; proteolyticor catalytic activity such as to effect factor X (FX) activation;antigenicity (ability to bind to or compete with a polypeptide forbinding to an anti-FIX antibody); ability to bind FVIIIa, antithrombinIII, heparin and/or factor X; ability to bind to phospholipids;three-dimensional structure; pI; and/or conformation. Included among themodified FIX polypeptides provided herein are those that have increasedresistance to antithrombin III (AT-III), increased resistance toheparin, altered glycosylation, such as increased glycosylation,increased catalytic activity, and improved pharmacokinetic properties,such as i) decreased clearance, ii) altered volume of distribution, iii)enhanced in vivo recovery, iv) enhanced total protein exposure in vivo(i.e., AUC), v) increased serum half-life (α-, β-, and/or γ-phase),and/or vi) increased mean resonance time (MRT).

In some examples, a modification can affect two or more properties oractivities of a FIX polypeptide. For example, a modification can resultin increased AT-III resistance and increased catalytic activity of themodified FIX polypeptide compared to an unmodified FIX polypeptide. Inanother example, a modification that introduces a non-nativeN-glycosylation site and, thus, can increase the glycosylation levels ofthe polypeptide when expressed in an appropriate cell, such as amammalian cell, also can result in increased catalytic activity of themodified FIX polypeptide compared to an unmodified FIX polypeptide.Modified FIX polypeptides provided herein can be assayed for eachproperty and activity to identify the range of effects of amodification. Such assays are known in the art and described below.Typically, changes to the properties and/or activities of the modifiedFIX polypeptides provided herein are made while retaining other FIXactivities or properties, such as, but not limited to, binding to FVIIIaand/or binding and activation of FX. Hence, modified FIX polypeptidesprovided herein retain FVIIIa binding and/or FX binding and activationas compared to a wild-type or starting form of the FIX polypeptide.Typically, such activity is substantially unchanged (less than 1%, 5% or10% changed) compared to a wild-type or starting protein. In otherexamples, the activity of a modified FIX polypeptide is increased or isdecreased as compared to a wild-type or starting FIX polypeptide.Activity can be assessed in vitro or in vivo and can be compared to theunmodified FIX polypeptide, such as for example, the mature, wild typenative FIX polypeptide (SEQ ID NO:3), the wild-type precursor FIXpolypeptide (SEQ ID NO:2), or any other FIX polypeptide known to one ofskill in the art that is used as the starting material.

The modifications provided herein can be made by standard recombinantDNA techniques such as are routine to one of skill in the art. Anymethod known in the art to effect mutation of any one or more aminoacids in a target protein can be employed. Methods include standardsite-directed mutagenesis of encoding nucleic acid molecules, or bysolid phase polypeptide synthesis methods.

Other modifications that are or are not in the primary sequence of thepolypeptide also can be included in a modified FIX polypeptide, orconjugate thereof, including, but not limited to, the addition of acarbohydrate moiety, the addition of a polyethylene glycol (PEG) moiety,the addition of an Fc domain, a serum albumin and/or other protein. Forexample, such additional modifications can be made to increase thestability or half-life of the protein.

The resulting modified FIX polypeptides include those that aresingle-chain zymogen polypeptides and those that are two-chainzymogen-like polypeptides (i.e. FIXa polypeptides that are not bound tothe cofactor, FVIIIa). Any modified FIX polypeptide provided herein thatis a single-chain polypeptide can be activated to generate a modifiedFIXa (i.e. a two-chain form). The activities of a modified FIXpolypeptide are typically exhibited in its two-chain form.

1. Exemplary Amino Acid Replacements

Provided herein are modified FIX polypeptides that contain one or moreamino acid replacements as described herein below with numbering ofresidues with respect to the numbering of SEQ ID NO:3. The same aminoacid replacements can be made in corresponding amino acid residues inanother FIX polypeptide (see e.g. FIGS. 3A-3D for exemplification ofidentification of corresponding amino acid residues). The amino acidreplacements confer altered glycosylation (e.g. by introduction ofnon-native glycosylation sites or elimination of native glycosylationsites), increased resistance to AT-III and/or heparin, increasedcatalytic activity, decreased LRP binding and/or alteredposttranslational modifications. The resulting modified FIX polypeptidesexhibit improved therapeutic efficacy, for example, due to improvedpharmacodynamic or pharmacokinetic activity.

In particular, non-limiting examples of amino acid replacements inmodified FIX polypeptides provided herein below are at any one or moreamino acid residues 155, 318, 338, 343, 403 and/or 410 with numberingwith respect to the mature FIX polypeptide set forth in SEQ ID NO:3(corresponding to amino acid residues [155], 150, 170, 175, 233 and/or240, respectively, by chymotrypsin numbering). The residuescorresponding to any of 155, 318, 338, 343, 403 and/or 410 in other FIXpolypeptides can be determined by sequence alignment with SEQ ID NO:3(see e.g. FIGS. 3A-3D). It is understood that the amino acidreplacements provided herein at any of amino acid residues 155, 318,338, 343, 403 and/or 410 with numbering with respect to SEQ ID NO:3 canbe made in other FIX polypeptides as described elsewhere herein. It isalso understood that residues corresponding to any of the other aminoacid replacements provided herein also can be identified in other FIXpolypeptides as exemplified herein (e.g. FIGS. 3A-3D).

In particular, provided herein are amino acid replacements of tyrosineat amino acid residue Y155 (Y155F), Y155L, Y155H, R318A, R318Y, R318E,R318F, R318W, R318D, R3181, R318K, R318L, R318M, R318N, R318S, R318V,R318Y, R338A, R338E, T343R, T343E, T343D, T343F, T343I, T343K, T343L,T343M, T343Q, T343S, T343V, T343W, T343Y, R403A, R403E, E410Q, E410S,E410N, E410A, E410D, or a conservative amino acid replacement (see e.g.Table 2B). In some examples, the amino acid replacement is Y155F, R318Y,R318E, R338E, T343R, R403E and/or E410N or conservative amino acidreplacements thereof.

For example, as shown by the data herein, amino acid replacement atposition R318 with reference to SEQ ID NO:3 (150 by chymotrypsinnumbering) confers resistance to inhibition by the AT-III/heparincomplex. An amino acid replacement at position R338 (R170 bychymotrypsin numbering) also confers resistance to inhibition by theAT-III/heparin complex. In this respect, the amino acid position R338 isthe site of a natural mutation (R170L) that has been reported to exhibit5-10 fold enhanced clotting activity in an in vitro clotting assay(International Pat. Pub. No. WO 2010029178). The assay as described wasperformed with conditioned media rather than purified protein and theprotein concentration was measured using an ELISA assay. Consequently,these data could reflect a higher fraction of active material in theR338L (R170L) preparation as compared to the wildtype comparatorpreparation or a higher level of contaminants that are active in aclotting assay. Nevertheless, as shown herein, there is a 3.5- to 4-foldincreased efficiency for FX activation by variants containing A, E and Lat position 338 (170). As found herein, the R338E mutation, in addition,exhibited an approximately 88-fold resistance to inhibition by theheparin/AT-III complex as well as 2-fold enhanced binding to theco-factor, FVIIIa.

A 4 amino acid thrombin loop swap mutation into FIX, from positions342-345 (174-177 by chymotrypsin numbering) has been reported to reducethe binding of FIXa to sLRP (see, Rohlena et al., (2003) J. Biol. Chem.9394-9401). Mutation of the residue at position T343 (T175 bychymotrypsin numbering) did not confer any significant affect on thepharmacokinetic (PK) properties of FIX. It is found herein that themutation T343R (T175R by chymotrypsin numbering), however, increases thecatalytic efficacy for activation of FX by a factor of about 3.1,produces an approximately 5.6-fold resistance to the heparin/AT-IIIcomplex, and increases the affinity for FVIIIa by a factor ofapproximately 1.6-fold.

Also as shown herein, mutations at position R403 (R233 by chymotrypsinnumbering) confer resistance to inhibition by the heparin/AT-IIIcomplex. Mutations at position E410 (E240 by chymotypsin numbering),such as E410N, produce a significant, heretofore unobserved, 1.3- to2.8-fold increase in the catalytic efficacy for activation of FX.

Also, as shown therein, there is a synergy in mutations at R338 and T343(R170 and T175 by chymotrypsin numbering), particularly R338E and T343Rin enhanced binding to the co-factor FVIII. Synergy also was observedbetween mutations at positions R338 and E410 (R170 and E240 bychymotrypsin numbering), particularly R338E and E410N. The two doublemutants, exemplified herein, R338E/T343R and R338E/E410N exhibit 24- to28-fold improved binding to FVIIIa while each of the single mutationsalone enhance binding by 1.6-2.2-fold each.

Other exemplary amino acid replacements in a FIX polypeptide providedherein found to confer an altered property or activity as describedbelow can be at any amino acid residue from among 1, 5, 53, 61, 64, 85,103, 104, 105, 106, 108, 148, 157, 158, 159, 167, 169, 172, 179, 202,203, 204, 205, 228, 239, 241, 243, 247, 249, 251, 257, 259, 260, 262,284, 293, 312, 314, 315, 316, 317, 319, 320, 321, 333, 342, 345, 346,392, 394, 400, 412, or 413 with reference to SEQ ID NO:3 or at acorresponding amino acid residue. For example, exemplary amino acidreplacements in a FIX polypeptide provided herein also include, but arenot limited to, Y1N, K5A, S53A, S61A, S61C, S61D, S61E, S61F, S61G,S61I, S61K, 561L, S61P, S61R, S61V, S61W, 561Y, D64A, D64C, D64F, D64H,D64I, D64L, D64M, D64N, D64P, D64R, D64S, D64T, D64W, D85N, A103N,D104N, N105S, N105T, K106N, K106S, K106T, V108S, V108T, T148A, N157D,N157E, N157F, N157I, N157K, N157L, N157M, N157Q, N157R, N157V, N157W,N157Y, S158A, S158D, S158E, S158F, S158G, S158I, S158K, 5158L, S158M,S158R, S158V, S158W, S158Y, T159A, N167D, N167Q, N167E, N167F, N167G,N167H, N167I, N167K, N167L, N167M, N167P, N167R, N167V, N167W, N167Y,T169A, T169D, T169E, T169F, T169G, T169I, T169K, T169L, T169M, T169P,T169R, T169S, T169V, T169W, T169Y, T172A, T172D, T172E, T172F, T172G,T172I, T172K, T172L, T172M, T172P, T172R, T172S, T172V, T172W, T172Y,T179A, V202M, V202Y, D203N, D203M, D203Y, D203F, D203H, D203I, D203K,D203L, D203R, D203V, D203W, A204M, A204Y, A204F, A204I, A204W, F205S,F205T, K228N, E239A, E239S, E239R, E239K, E239D, E239F, E239I, E239L,E239M, E239N, E239T, E239V, E239W, E239Y, T241N, H243S, H243T, K247N,N249S, N249T, 1251S, H257F, H257E, H257D, H257I, H257K, H257L, H257M,H257Q, H257R, H257S, H257V, H257W, H257Y, N260S, A262S, A262T, Y284N,K293E, K293A, R312A, R312Y, R312L, R312C, R312D, R312E, R312F, R312I,R312K, R312L, R312M, R312P, R312Q, R312S, R312T, R312V, R312W, R312Y,F314N, H315S, K316M, K316D, K316F, K316H, K316I, K316L, K316M, K316R,K316S, K316T, K316V, K316W, K316Y, G317N, S319N, A320S, L321N, L321S,L321T, R333A, R333E, F342I, F342D, F342E, F342K, F342L, F342M, F342S,F342T, F342V, F342W, F342Y, Y345A, Y345T, N346D, N346Y, N346E, N346F,N346H, N346I, N346K, N346L, N346M, N346Q, N346R, N346T, N346V, N346W,K392N, K394S, K394T, K400A, K400E, K400C, K400D, K400F, K400G, K400L,K400M, K400P, K400S, K400T, K400V, K400Y, T412A, T412V, T412C, T412D,T412E, T412F, T412G, 1412I, T412M, T412P, T412W, T412Y, K413N in amature FIX polypeptide having a sequence set forth in SEQ ID NO:3 or thesame replacement in a corresponding amino acid residue position.

For example, exemplary properties and activies that are altered by themodifications (e.g. amino acid replacements) provided herein aredescribed as follows.

a. Altered Glycosylation

The modified factor IX polypeptides provided herein can exhibit alteredglycosylation levels and/or altered types of glycosylation compared toan unmodified FIX polypeptide. In some examples, the modified FIXpolypeptides provided herein exhibit increased glycosylation compared toan unmodified FIX polypeptide. Thus, among the modified FIX polypeptidesdescribed herein are hyperglycosylated FIX polypeptides.

i. Advantages of Glycosylation

Many mammalian proteins are glycosylated with variable numbers ofcarbohydrate chains, each of which can have differing carbohydratestructures. Such carbohydrates can have an important role in thestability, solubility, activity, serum half-life and immunogenicity ofthe protein. Thus, the properties and activities of a protein can bealtered by modulating the amount and/or type of glycosylation. Forexample, glycosylation can increase serum-half-life of polypeptides byincreasing the stability, solubility, and reducing the immunogenicity ofa protein. This is of particular interest for therapeutic polypeptides,where increased solubility, serum half-life and stability of thetherapeutic polypeptide can result in increased therapeutic efficacy.

Oligosaccharides are important in intra- and inter-cell events such as arecognition, signaling and adhesion. Carbohydrates also assist in thefolding of secreted proteins. Glycosylation sites provide a site forattachment of monosaccharides and oligosaccharides to a polypeptide viaa glycosidic linkage, such that when the polypeptide is produced, forexample, in a eukaryotic cell capable of glycosylation, it isglycosylated. There are several types of protein glycosylation. N-linkedand O-linked glycosylation are the major classes, in which an asparagineresidue, or a serine or threonine residue, respectively, is modified.Other types of glycans include, glycosaminoglycans andglycosylphophatidylinositol (GPI)-anchors. Glycosaminoglycans areattached to the hydroxy oxygen of serine, while GPI anchors attach aprotein to a hydrophobic lipid anchor, via a glycan chain.C-glycosylation also can occur at the consensus sequence Trp-X-X-Trp,where the indol side chain of the first tryptophan residue in thesequences is modified with an α-mannopyranosyl group (Furmanek et al.,(2000) Acta Biochim. Pol. 47:781-789).

The presence of a potential glycosylation site does not, however, ensurethat the site will be glycosylated during post-translational processingin the ER. Furthermore, the level of glycosylation can vary at any givensite, as can the glycan structures. The differences in levels and typesof glycosylation at particular sites can be attributed, at least inpart, to the sequence context and secondary structure around thepotential glycosylation site.

O-linked glycosylation involves the attachment of the sugar units, suchas N-acetylgalactosamine, via the hydroxyl group of serine, threonine,hydroxylysine or hydroxyproline residues. It is initiated by theattachment of one monosaccharide, following which others are added toform a mature O-glycan structure. There is no known motif forO-glycosylation, although O-glycosylation is more probable in sequenceswith a high proportion of serine, threonine and proline residues.Further, secondary structural elements such as an extended β turn alsomay promote O-glycosylation. O-glycosylation lacks a common corestructure. Instead, several types of glycans can be attached at theselected O-glycosylation sites, including O—N-acetylgalactosamine(O-GalNAc), O—N-acetylglucosamine (O-GlcNAc), O-fucose and O-glucose.

In contrast to O-glycosylation, the N-linked glycosylation consensussequence motif is well characterized. During N-linked glycosylation, a14-residue oligosaccharide is transferred to the asparagine residue inthe Asn-X-Ser/Thr/Cys consensus motif, where X is any amino acid exceptPro. Glycosyltransferases then enzymatically trim the saccharide andattach additional sugar units to the mannose residues. The sequenceadjacent to the consensus motif also can affect whether or notglycosylation occurs at the consensus sequence. Thus, the presence ofthe Asn-X-Ser/Thr/Cys consensus sequence is required but not necessarilysufficient for N-linked glycosylation to occur. In some instances,changes to the adjacent sequence results in glycosylation at theconsensus motif where there previously was none (Elliot et al., (2004)J. Biol. Chem. 279:16854-16862).

N-linked oligosaccharides share a common core structure of GlcNAc₂Man₃.There are three major types of N-linked saccharides in mammals:high-mannose oligosaccharides, complex oligosaccharides and hybridoligosaccharides. High-mannose oligosaccharides essentially contain twoN-acetylglucosamines with several mannose residues. In some instances,the final N-linked high-mannose oligosaccharide contains as many mannoseresidues as the precursor oligosaccharide before it is attached to theprotein. Complex oligosaccharides can contain almost any number ofmannose, N-acetylglucosamines and fucose saccharides, including morethan the two N-acetylglucosamines in the core structure.

Glycosylation can increase the stability of proteins by reducing theproteolysis of the protein and can protect the protein from thermaldegradation, exposure to denaturing agents, damage by oxygen freeradicals, and changes in pH. Glycosylation also can allow the targetprotein to evade clearance mechanisms that can involve binding to otherproteins, including cell surface receptors. The sialic acid component ofcarbohydrate in particular can enhance the serum half-life of proteins.Sialic acid moieties are highly hydrophilic and can shield hydrophobicresidues of the target protein. This increases solubility and decreasesaggregation and precipitation of the protein. Decreased aggregationreduces the likelihood of an immune response being raised to theprotein. Further, carbohydrates can shield immunogenic sequences fromthe immune system, and the volume of space occupied by the carbohydratemoieties can decrease the available surface area that is surveyed by theimmune system. These properties can lead to the reduction inimmunogenicity of the target protein.

Modifying the level and/or type of glycosylation of a therapeuticpolypeptide can affect the in vivo activity of the polypeptide. Byincreasing the level of glycosylation, recombinant polypeptides can bemade more stable with increased serum half-life, reduced serum clearanceand reduced immunogenicity. This can increase the in vivo activity ofthe polypeptide, resulting in reduced doses and/or frequency of dosingto achieve a comparable therapeutic effect. For example, ahyperglycosylated form of recombinant human erythropoietin (rHuEPO),called Darbepoetin alfa (DA), has increased in vivo activity andprolonged duration of action. The increased carbohydrate and sialic acidcontent of the hyperglycosylated DA polypeptide results in a serumhalf-life that is three times greater than that of the unmodifiedrHuEPO. This increased serum half-life results in increasedbioavailability and reduced clearance, which can allow for less frequentdosing and/or lower dosages, with associated increased convenience forthe patient, reduced risk of adverse effects and improved patientcompliance.

ii. Exemplary Modified FIX Polypeptides with Altered Glycosylation

Provided herein are modified FIX polypeptides that are modified toexhibit altered glycosylation compared to an unmodified FIX polypeptide.The modified FIX polypeptides can exhibit increased or decreasedglycosylation, such as by the incorporation of non-native glycosylationsites or the deletion of native glycosylation sites, respectively. Forexample, the modified FIX polypeptides can contain 1, 2, 3, 4 or morenon-native N-glycosylation sites. The non-native N-glycosylation sitescan be introduced by amino acid replacement(s) (or substitution(s)),insertion(s) or deletion(s), or any combination thereof, wherein theamino acid replacement(s), insertion(s) and/or deletion(s) result in theestablishment of the glycosylation motif Asn-Xaa-Ser/Thr/Cys, where Xaais not proline. In other examples, the modified FIX polypeptidesprovided herein can have a reduced number of glycosylation sitescompared to an unmodified FIX polypeptide, typically resulting in areduced level of glycosylation compared to the unmodified FIXpolypeptide. In further examples, the modified FIX polypeptides exhibitthe same levels of glycosylation as wild-type FIX, but exhibit differenttypes of glycosylation. For example, a modified FIX polypeptide canexhibit the same number of glycosylation sites and the same level ofglycosylation as an unmodified FIX polypeptide, but can have differenttypes of glycosylation, such as, for example, different relative amountsof N- and O-glycosylation compared to an unmodified FIX polypeptide.

(a). Introduction of Non-Native Glycosylation Site(s)

In particular examples, a non-native N-glycosylation site is introducedby amino acid replacement. In some instances, the creation of anon-native N-glycosylation site by amino acid replacement requires onlyone amino acid replacement. For example, if the unmodified FIXpolypeptide contains a Gly-Ala-Ser sequence, then an N-glycosylationsite can be created by a single amino acid substitution of the glycinewith an asparagine, to create an Asn-Ala-Ser N-glycosylation motif. Inanother example, if the unmodified FIX polypeptide contains anAsn-Trp-Met sequence, then an N-glycosylation site can be created by asingle amino acid substitution of the methionine with a cysteine (orthreonine or serine). In other instances, the creation of a non-nativeN-glycosylation site by amino acid replacement requires more than oneamino acid replacement. For example, if the unmodified FIX polypeptidecontains a Gly-Arg-Phe sequence, then an N-glycosylation site can becreated by two amino acid replacements: an amino acid substitution ofthe glycine with an asparagine, and an amino acid substitution of thephenylalanine with a cysteine (or threonine or serine), to create aAsn-Arg-Ser/Thr/Cys N-glycosylation motif. Thus, one of skill in the artcan introduce one or more non-native N-glycosylation sites at anyposition in the FIX polypeptide.

The position at which a non-native glycosylation site is introduced intothe FIX polypeptide to generate the modified FIX polypeptides providedherein is typically selected so that any carbohydrate moieties linked atthat site do not adversely interfere with the structure, function and/orprocoagulant activity of the FIX polypeptide, or that the amino acidmodification(s) made to the polypeptide to introduce the non-nativeglycosylation site do not adversely interfere with the structure,function or activity of the FIX polypeptide. Thus, a non-nativeglycosylation site can be introduced into any position in a FIXpolypeptide provided the resulting modified FIX polypeptide retains atleast one activity of the wild type or unmodified FIX polypeptide.Conversely, one or more non-native glycosylation sites can be introducedinto the modified FIX polypeptide at sites that may be involved in theinteraction of FIX with an inhibitory molecule. The carbohydrate moietythat is linked to the new glycosylation site can sterically hinder theinteraction between the inhibitory molecule and the modified FIX. Suchsteric hindrance can result in a modified FIX polypeptide with increasedcoagulant activity. For example, a carbohydrate moiety that is linked toa non-native glycosylation site contained in the modified FIXpolypeptides provided herein can sterically hinder the interaction ofthe modified FIX with the AT-III/heparin complex. This can result inincreased resistance of the modified FIX polypeptide to the inhibitoryeffects of AT-III/heparin.

Thus, a non-native glycosylation site can be introduced into the Gladomain, EGF1 domain, EGF2 domain, activation peptide and/or the proteasedomain, provided the resulting modified FIX polypeptide retains at leastone activity of the wild type or unmodified FIX polypeptide. In otherexamples, a non-native glycosylation site is introduced into the EGF2domain or the protease domain. The resulting modified FIX polypeptideretains at least one activity of the unmodified FIX polypeptide. In someexamples, the modified FIX polypeptide retains at least 5%, 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more of the catalytic activityof the unmodified FIX polypeptide. In other examples, the modified FIXpolypeptide retains at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 95% or more of the binding activity for FX of the unmodified FIXpolypeptide. In other examples, the modified FIX polypeptide retains atleast 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more ofthe binding activity for FVIIIa of the unmodified FIX polypeptide. Insome assays and/or under some conditions, the modified FIX polypeptidescan exhibit increased activity compared with the unmodified FIX protein(e.g., pharmacodynamic activity in vivo, and/or catalytic activity inthe presence of ATIII/heparin or plasma)

Table 3 provides non-limiting examples of exemplary amino acidreplacements, corresponding to amino acid positions of a mature FIXpolypeptide as set forth in SEQ ID NO:3, that are included in a modifiedFIX polypeptide to increase glycosylation levels by introducing anon-native N-glycosylation site. In reference to such mutations, thefirst amino acid (one-letter abbreviation) corresponds to the amino acidthat is replaced, the number corresponds to the position in the matureFIX polypeptide sequence with reference to SEQ ID NO:3, and the secondamino acid (one-letter abbreviation) corresponds to the amino acidselected that replaces the first amino acid at that position. The aminoacid positions for mutation also are referred to by the chymotrypsinnumbering scheme where appropriate (i.e., when the mutation is locatedwithin the FIX protease domain). In instances where a modified aminoacid position does not have a corresponding chymotrypsin number (i.e. isnot within amino acid positions 181 to 415 corresponding to a mature FIXpolypeptide set forth in SEQ ID NO:3, and is not set forth in Table 1,above), the position is denoted in brackets using mature FIX numbering.For example, A103N does not have a corresponding chymotrypsin number andis set forth as A[103]N when referring to chymotrypsin numbering. InTable 3 below, the sequence identifier (SEQ ID NO) is identified inwhich exemplary amino acid sequences of the modified FIX polypeptide areset forth. Also identified in Table 3 are the positions of thenon-native glycosylation sites generated by the modifications.

In some instances, only one amino acid replacement is required to createa non-native N-glycosylation site. For example, the aspartic acid (Asp,D) at position 85 (corresponding to the mature FIX polypeptide set forthin SEQ ID NO:3) can be replaced with an asparagine (Asn, N) to generatea non-native glycosylation site in the EGF2 domain at amino acidposition 85 in the resulting modified FIX polypeptide. In anotherexample, the isoleucine (Ile, I) at position 251 (corresponding to themature FIX polypeptide set forth in SEQ ID NO:3) can be replaced with aserine (Ser, S) to generate a non-native N-glycosylation site in theprotease domain at amino acid position 249 in the resulting modified FIXpolypeptide. In other instances, two amino acid replacements arerequired to create a new glycosylation site. For example, the alanine(Ala, A) at position 103 (based on numbering of a mature FIX set forthin SEQ ID NO:3) can be replaced with an asparagine (Asn, N), and theasparagine at position 105 can be replaced with a serine (Ser, S) tocreate a non-native N-glycosylation site in the EGF2 domain at aminoacid position 103 in the resulting modified FIX polypeptide. In anotherexample, the threonine (Thr, T) at position 241 is replaced with anasparagine and the histidine (His, H) at position 243 is replaced with aserine to create a non-native N-glycosylation site in the proteasedomain at amino acid position 243.

TABLE 3 Non-native Non-native glycosylation glycosylation ModificationModification site site SEQ (mature FIX (chymotrypsin (mature FIX(chymotrypsin ID numbering) numbering) numbering) numbering) NOA103N/N105S A[103]N/N[105]S N103 N[103] 77 D104N/K106S D[104]N/K[106]SN104 N[104] 78 K106N/V108S K[106]N/V[108]S N106 N[106] 79 D85N D[85]NN85 N[85] 80 D203N/F205T D39N/F41T N203 N39 99 K228N K63N N228 N63 101I251S I86S N249 N84 103 A262S A95bS N260 N95 106 K413N K243N N413 N243107 E410N E240N N410 N240 108 E239N E74N N239 N74 109 T241N/H243ST76N/H78S N241 N76 110 K247N/N249S K82N/N84S N247 N82 111 L321N L153NN321 N153 112 K392N/K394S K222N/K224S N392 N222 114 N260S N95S N258 N93116 S319N/L321S S151N/L153S N319 N151 115 Y284N Y117N N284 N117 117G317N G149N N317 N149 118 R318N/A320S R150N/A152S N318 N150 119F314N/K316S F145N/K148S N314 N145 177

The modified FIX polypeptides provided herein can contain modificationsthat result in the introduction of two or more non-nativeN-glycosylation sites. For example, the modifications set forth in Table3 can be combined, resulting in a modified FIX polypeptide that contains2, 3, 4, 5, 6 or more non-native N-glycosylation sites. Any two or moreof the modifications set forth in Table 3 can be combined. For example,included among the modified FIX polypeptides provided herein aremodified FIX polypeptides that contain the amino acid substitutionsD104N/K106S/K228N, resulting in a FIX polypeptide with two non-nativeglycosylation sites at amino acid positions 104 and 228, respectively(numbering corresponding to the mature FIX polypeptide set forth in SEQID NO:3). In another example, a modified FIX polypeptide can containamino acid substitutions D85N/K247N/N249S/K392N/K394S, resulting in aFIX polypeptide with three non-native glycosylation sites at amino acidpositions 85, 247 and 392, respectively (numbering corresponding to themature FIX polypeptide set forth in SEQ ID NO:3). Table 4 sets forthexemplary FIX polypeptides having two or more non-native N-glycosylationsites.

TABLE 4 Non-native Non-native glycosylation glycosylation ModificationsModifications site site SEQ (mature FIX (chymotrypsin (mature FIX(chymotrypsin ID numbering) numbering) numbering) numbering) NO.D85N/I251S D[85]N/I86S N85 and N149 N[85] and N84 104 D85N/D203N/F205TD[85]N/D39N/F41T N85 and N203 N[85] and N39 100 D85N/K228N D[85]N/K63NN85 and N228 N[85] and N63 102 D85N/D104N/ D[85]N/D[104N]/ N85, N104N[85], N[104] 105 K106S/I251S K[106]6/I86S and N249 and N84 A103N/N105S/A[103]N/N[105]S/ N103 and N[103] and 178 K247N/N249S K82N/N84S N247 N82D104N/K106S/ D[104]N/K[106]S/ N104 and N[104] and 179 K247N/N249SK82N/N84S N247 N82 K228N/I251S K63N/I86S N228 and N63 and N84 180 N249A103N/N105S/I251S A[103]N/N[105]S/I86S N103 and N[103] and 181 N249 N84D104N/K106S/I251S D[104]N/K[106]S/I86S N104 and N[104] and 182 N249 N84K228N/K247N/N249S K63N/K82N/N84S N228 and N63 and N82 183 N247K228N/K247N/N249S/ K63N/K82N/N84S/ N228, N247 N63, N82 and 184D104N/K106S D[104]N/K[106]S and N104, N[104] D104N/K106S/N260SD[104]N/K[106]S/N95S N104 and N[104] and 185 N258 N93

The modified FIX polypeptides provided herein can contain one or morenon-native glycosylation sites, such as one or more non-nativeN-glycosylation sites. Thus, when expressed in a cell that facilitatesglycosylation, or when glycosylated using in vitro techniques well knowin the art, the modified FIX polypeptides can exhibit increased levelsof glycosylation compared to an unmodified FIX polypeptide. The level ofglycosylation can be increased by at least or at least about 1%, 2%, 3%,4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,100%, 200%, 300%, 400%, 500%, or more compared to the level ofglycosylation of unmodified or wild-type FIX polypeptide.

The modifications described herein to introduce one or more non-nativeglycosylation sites can be combined with any other mutation describedherein or known in the art. Typically, the resulting modified FIXpolypeptide exhibits increased coagulant activity compared to anunmodified FIX polypeptide. For example, one or more modifications thatintroduce one or more non-native glycosylation sites can be combinedwith modification(s) that increase resistance to an inhibitor, such asAT-III and/or heparin, increase catalytic activity, increase intrinsicactivity, increase binding to phospholipids, decrease binding to LRPand/or improve pharmacokinetic and/or pharmacodynamic properties.

The modified FIX polypeptides provided herein that contain one or morenon-native glycosylation sites and have altered glycosylation, such asincreased levels of glycosylation, retain at least one activity of FIX,such as, for example, catalytic activity for its substrate, FX.Typically, the modified FIX polypeptides provided herein retain at leastor at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%or more of the catalytic activity exhibited by an unmodified FIXpolypeptide. Increased levels of glycosylation can improve thepharmacokinetic properties of the modified FIX polypeptides by endowingthe variant with one or more of the following properties: i) decreasedclearance, ii) altered volume of distribution, iii) enhanced in vivorecovery, iv) enhanced total protein exposure in vivo (i.e., AUC), v)increased serum half-life (α, β, and/or γ phase), and/or vi) increasedmean resonance time (MRT) compared to an unmodified FIX. The coagulantactivity of the modified FIX polypeptides with altered glycosylation canbe increased by at least or at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%,400%, 500%, or more compared to the coagulation activity of unmodifiedor wild-type FIX polypeptide either in vivo or in vitro.

(b). Elimination of Native Glycosylation Sites

The modified FIX polypeptides provided herein can have a reduced numberof glycosylation sites compared to an unmodified FIX polypeptide.Typically, a reduction in the number of glycosylation sites results in areduced level of glycosylation compared to the unmodified FIXpolypeptide. The native glycosylation sites that can be removed include,for example, native N-glycosylation sites at amino acid positionscorresponding to positions 157 and 167 of the mature FIX set forth inSEQ ID NO:3, and native O-glycosylation sites at amino acid positionscorresponding to positions 53, 61, 159, 169, 172 and 179 of the matureFIX set forth in SEQ ID NO:3.

Any one or more native glycosylation sites can be removed by amino acidreplacement(s), insertion(s) or deletion(s), or any combination thereof.For example, an amino acid replacement, deletion and/or insertion can bemade to destroy the Asn/Xaa/Ser/Thr/Cys motif (where Xaa is not aproline), thereby removing an N-glycosylation site at position 157 or167. In other examples, O-glycosylation sites are removed, such as byamino acid replacement or deletion of the serine residues at positions53 or 61, or amino acid replacement or deletion of the threonineresidues at positions 159 or 169. The resulting modified FIX polypeptideretains at least one activity of the unmodified FIX polypeptide. In someexamples, the modified FIX polypeptide retains at least 5%, 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more of the catalytic activityof the unmodified FIX polypeptide. In other examples, the modified FIXpolypeptide retains at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 95% or more of the binding activity for FX of the unmodified FIXpolypeptide. In other examples, the modified FIX polypeptide retains atleast 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more ofthe binding activity for FVIIIa of the unmodified FIX polypeptide. Insome assays and/or under some conditions, the modified FIX polypeptidescan exhibit enhanced properties compared with unmodified FIX (e.g.,including but not limited to, increased in vivo recovery, increased AUCin vivo, and/or decreased clearance in vivo).

Table 5 provides non-limiting examples of exemplary amino acidreplacements, corresponding to amino acid positions of a mature FIXpolypeptide as set forth in SEQ ID NO:3, that are included in a modifiedFIX polypeptide to decrease glycosylation levels by removing oreliminating a native N-glycosylation site. In Table 5 below, thesequence identifier (SEQ ID NO) is identified in which exemplary aminoacid sequences of the modified FIX polypeptide are set forth.

TABLE 5 Mutation Mutation SEQ ID (Mature FIX Numbering) (ChymotrypsinNumbering) NO S53A S[53]A 88 S61A S[61]A 87 N157D N[157]D 75 N157QN[157]Q 98 T159A T[159]A 89 N167D N[167]D 85 N167Q N[167]Q 86 T169AT[169]A 90 T172A T[172]A 91 T179A T[179]A 92

The modifications described herein to eliminate one or more nativeglycosylation sites can be combined with any other mutation describedherein or known in the art. Typically, the resulting modified FIXpolypeptide exhibits increased coagulant activity compared to anunmodified FIX polypeptide. For example, one or more modifications thateliminate one or more native glycosylation sites can be combined withmodification(s) that introduce a non-native glycosylation site, increaseresistance to an inhibitor, such as AT-III and/or heparin, increasecatalytic activity, increase intrinsic activity, increase binding tophospholipids, or improve pharmacokinetic and/or pharmacodynamicproperties.

The modified FIX polypeptides provided herein that eliminate one or morenative glycosylation sites retain at least one activity of FIX, such as,for example, catalytic activity for its substrate, FX. Typically, themodified FIX polypeptides provided herein retain at least or at leastabout 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more ofthe catalytic activity exhibited by an unmodified FIX polypeptide. Insome instances, the coagulant activity of the modified FIX polypeptideswith altered glycosylation can be increased by at least or at leastabout 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more compared to thecoagulation activity of unmodified or wild-type FIX polypeptide eitherin vivo or in vitro.

b. Increased Resistance to AT-III and Heparin

The activity of FIX can be inhibited by factors in the blood as part ofthe regulation of the coagulation process. Thus, provided herein aremodified FIX polypeptides that exhibit increased resistance to theinhibitory effects of inhibitors, including AT-III and heparin. In someexamples, the modified FIX polypeptides provided herein exhibit reducedbinding affinity for heparin and/or a decreased second order rateconstant for inhibition by AT-III alone and/or the AT-III/heparincomplex. In further examples, the modified FIX polypeptides exhibitincreased resistance to the AT-III alone, or heparin alone. Thus,provided herein are modified FIX polypeptides that exhibit increasedresistance to AT-III, the AT-III/heparin complex and/or heparin.

i. AT-III

Antithrombin III (also known as antithrombin or AT-III) is an importantanticoagulant serpin (serine protease inhibitor). AT-III is synthesizedas a precursor protein containing 464 amino acid residues (SEQ IDNO:21). In the course of secretion a 32 residue signal peptide iscleaved to generate a 432 amino acid mature human antithrombin (SEQ IDNO:22). The 58 kDa AT-III glycoprotein circulates in the blood andfunctions as a serine protease inhibitor (serpin) to inhibit a largenumber of serine proteases of the coagulation system. The principaltargets of AT-III are thrombin, factor Xa and factor IXa, althoughAT-III also has been shown to inhibit the activities of FXIa, FXIIa and,to a lesser extent, FVIIa.

The action of AT-III is greatly enhanced by glycosaminoglycans, such asthe naturally occurring heparan sulphate or the various tissue-derivedheparins that are widely used as anticoagulants in clinical practice.Unlike other serpins, which typically are effective without binding asecondary molecule, the reaction of AT-III in the absence of heparinwith is target coagulations factors is unusually slow. In the absence ofheparin, the reactive loop sequence of AT-III provides the determinantsof the slow reactivity. Mutagenesis of the conserved P2-P1′ residues inthe reactive loop center of AT-III, for example, affects the interactionof AT-III with proteases in the absence but not the presence of heparin.

AT-III binds in a highly specific manner to a unique pentasaccharidesequence in heparin that induces a conformational change in the reactivecenter loop. In such a conformation, the reactive center loop of AT-IIIcan more efficiently interact with the reactive site of the serineprotease, and effect inhibition. Evidence suggests that binding ofheparin to AT-III generates new exosites that promote the interaction ofFIXa, thrombin and FXa with AT-III. The tyrosine at position 253 and theglutamic acid at position 255, for example, have been shown to be keydeterminants of an exosite on AT-III that is generated by heparinbinding, and that promotes the rapid, increased inhibition of FIXa byAT-III, compared to the inhibition observed with AT-III alone (Izaguirreet al., (2006) J. Bio Chem 281:13424-13432).

Mutational studies also have given some indication of which residues inFactor IXa are involved in the interaction with AT-III/heparin. Forexample, modification of the arginine at position 318 of the mature FIXpolypeptide (corresponding to position 150 by chymotrypsin numbering)reduces the reactivity of this mutant with AT-III/heparin by 33-fold to70-fold (Yang, L. et al., (2003) J. Biol. Chem. 278(27):25032-8). Theimpairment of the reactivity between the FIXa mutant and AT-III is notas noticeable when AT-III is not bound to heparin, however, indicatingthat the interaction between the arginine at position 318 of the matureFIXa polypeptide and AT-III is effected when AT-III is in theheparin-activated conformation.

ii. Heparin

Heparin can inhibit the activity of FIXa in the intrinsic tenase complexin both an AT-III-dependent manner, as discussed above, and anAT-III-independent manner. Studies indicate that the AT-III-independentinhibition of FIXa activity by heparin is the result of oligosaccharidebinding to an exosite on FIXa that disrupts the FVIIIa-FIXa interaction(Yuan et al., (2005) Biochem. 44:3615-3625, Misenheimer et al., (2007)Biochem. 46:7886-7895, Misenheimer et al. (2010) Biochem.49:9977-10005). The heparin-binding exosite is in the Factor IXaprotease domain, in an electropositive region extending from thearginine at position 338 (corresponding to position 170 by chymotrypsinnumbering) to at least the arginine at position 403 (corresponding toposition 233 by chymotrypsin numbering). This exosite overlaps with aregion of FIXa that is critical to the interaction of FIXa with itscofactor, FVIIIa. Thus, binding of heparin to FIXa inhibits theinteraction of FIXa with FVIIIa, thus reducing the intrinsic tenaseactivity.

iii. Exemplary FIX Polypeptides with Increased Resistance to AT-III andHeparin

Modifications can be made to a FIX polypeptide that increase itsresistance to AT-III, heparin and/or the AT-III/heparin complex.Generally, such modified FIX polypeptides retain at least one activityof a FIX polypeptide. Typically, such modifications include one or moreamino acid substitutions at any position of the FIX polypeptide that isinvolved in the interaction of FIXa with AT-III, heparin an/or theAT-III/heparin complex. Such modifications can, for example, result in areduced rate of interaction of the modified FIXa polypeptide with AT-IIIalone, a reduced rate of interaction of the modified FIXa polypeptide tothe AT-III/heparin complex, and/or a reduced binding affinity of themodified FIXa polypeptide for heparin alone. In some examples, themodification(s) introduces one or more non-native glycosylation sites.The carbohydrate moiety that is linked to the new glycosylation site cansterically hinder the interaction of the modified FIX with theAT-III/heparin complex, resulting in increased resistance of themodified FIX polypeptide to the inhibitory effects of AT-III/heparin.The modified FIXa polypeptides therefore exhibit increased resistance tothe naturally inhibitory effects of AT-III, AT-III/heparin and/orheparin with respect to intrinsic tenase activity. When evaluated in anappropriate in vitro assay, or in vivo, such as following administrationto a subject as a pro-coagulant therapeutic, the modified FIXpolypeptides display increased coagulant activity as compared withunmodified FIX polypeptides.

As described herein below, one of skill in the art can empirically orrationally design modified FIXa polypeptides that display increasedresistance to AT-III, AT-III/heparin and/or heparin. Such modified FIXpolypeptides can be tested in assays known to one of skill in the art todetermine if the modified FIX polypeptides display increased resistanceto AT-III, AT-III/heparin and/or heparin. For example, the modified FIXpolypeptides can be tested for binding to AT-III, AT-III/heparin and/orheparin. Generally, a modified FIX polypeptide that has increasedresistance to AT-III, AT-III/heparin and/or heparin will exhibitdecreased binding and/or decreased affinity for heparin and/or adecreased rate of interaction with AT-III and/or AT-III/heparin.Typically, such assays are performed with the activated form of FIX(FIXa), and in the presence or absence of the cofactor, FVIIIa, andphospholipids.

Provided herein are modified FIX polypeptides exhibiting increasedresistance to AT-III, AT-III/heparin and/or heparin. FIX polypeptidevariants provided herein have been modified at one or more of amino acidpositions 202, 203, 204, 205, 228, 239, 257, 260, 293, 312, 316, 318,319, 321, 333, 338, 342, 346, 400, 403 or 410 (corresponding to aminoacid positions 38, 39, 40, 41, 63, 74, 92, 95, 126, 143, 145, 148, 150,151, 153, 165, 170, 174, 178, 230, 233 and 240 respectively, bychymotrypsin numbering). These amino acid residues can be modified suchas by amino acid replacement, deletion or substitution. The identifiedresidues can be replaced or substituted with any another amino acid.Alternatively, amino acid insertions can be used to alter theconformation of a targeted amino acid residue or the protein structurein the vicinity of a targeted amino acid residue.

Any amino acid residue can be substituted for the endogenous amino acidresidue at the identified positions. Typically, the replacement aminoacid is chosen such that it interferes with the interaction between FIXand AT-III or heparin. For example, modifications can be made at aminoacid positions 260, 293, 333, 338, 346, 400 and 410 (corresponding toamino acid positions 95, 126, 165, 170, 178, 230, 233 and 240,respectively, by chymotrypsin numbering) to interfere with theinteraction of the FIX polypeptide with heparin. In other examples,modifications are made at amino acid positions 203, 204, 205, 228, 239,312, 314, 316, 318, 319, 321 and 342 (corresponding to amino acidpositions 39, 40, 41, 63, 74, 143, 145, 148, 150, 151, 153 and 174,respectively, by chymotrypsin numbering) to interfere with theinteraction of the FIX polypeptide with AT-III.

In some examples, a new glycosylation site is introduced by amino acidreplacement. The carbohydrate moiety that is linked to the newglycosylation site can sterically hinder the interaction of the modifiedFIX with the AT-III/heparin complex, resulting in increased resistanceof the modified FIX polypeptide to the inhibitory effects ofAT-III/heparin. For example, the glutamic acid (Glu, E) at position 410(corresponding to position 240 by chymotrypsin numbering) can bereplaced with an asparagine (Asn, N) to introduce a new glycosylationsite at position 410. In other examples, the glutamic acid (Glu, E) atposition 239 (corresponding to position 74 by chymotrypsin numbering) isreplaced with an asparagine (Asn, N) to introduce a new glycosylationsite at position 239. Other mutations that introduce a new glycosylationsite to increase resistance to AT-III/heparin include, for example,D203N/F205T, R318N/A320S, N260S and F314N/K316S (corresponding toD39N/F41T, R150N/A152S, N95S and F145N/K148S by chymotrypsin numbering).

In other examples in which modifications are made to increase resistanceto AT-III, AT-III/heparin and/or heparin, the valine residue at position202 (corresponding to position 38 by chymotrypsin numbering) is replacedwith a methionine (Met, M) or tyrosine (Tyr, Y); the aspartic acid (Asp,D) at position 203 (corresponding to position 39 by chymotrypsinnumbering) is replaced with a methionine (Met, M) or tyrosine (Tyr, Y);the alanine (Ala, A) at position 204 (corresponding to position 40 bychymotrypsin numbering) is replaced with a methionine (Met, M) ortyrosine (Tyr, Y); the glutamic acid at position 239 (corresponding toposition 74 by chymotrypsin numbering) is replaced with serine (Ser, S),alanine (Ala, A), arginine (Arg, R), or lysine (Lys, K); the histidineat position 257 (corresponding to position 92 by chymotrypsin numbering)is replaced with phenylalanine (Phe, F), tyrosine (Tyr, Y), glutamicacid (Glu, E) or serine (Ser, S); the lysine (Lys, K) at position 293(corresponding to position 143 by chymotrypsin numbering) is replacedwith alanine (Ala, A) or glutamine (Gln, Q); the arginine (Arg, R) atposition 312 (corresponding to position 143 by chymotrypsin numbering)is replaced with alanine (Ala, A) or glutamine (Gln, Q); the lysine atposition 316 (corresponding to 148 by chymotrypsin numbering) isreplaced with asparagine (Asn, N), alanine (Ala, A), glutamic acid (Glu,E), serine (Ser, S) or methionine (Met, M); the arginine (Arg, R) atposition 318 (corresponding to position 150 by chymotrypsin numbering)is replaced with alanine (Ala, A), glutamic acid (Glu, E) tyrosine (Tyr,Y), phenylalanine (Phe, F) or tryptophan (Trp, W); the arginine (Arg, R)at position 333 (corresponding to position 165 by chymotrypsinnumbering) is replaced with alanine (Ala, A) or glutamic acid (Glu, E);the arginine (Arg, R) at position 338 (corresponding to position 170 bychymotrypsin numbering) is replaced with alanine (Ala, A) or glutamicacid (Glu, E); the lysine (Lys, K) at position 400 (corresponding toposition 230 by chymotrypsin numbering) is replaced with alanine (Ala,A) or glutamic acid (Glu, E); and/or the arginine (Arg, R) at position403 (corresponding to position 233 by chymotrypsin numbering) isreplaced with alanine (Ala, A), glutamic acid (Glu, E) or aspartic acid(Asp, D).

Provided herein are modified FIX polypeptides that contain an amino acidreplacement at residue R318 or at a residue in a FIX polypeptidecorresponding to 318 that is a tyrosine, e.g., R318Y, or is aconservative amino acid replacement thereof. For example, conservativeamino acid residues for tyrosine include, but are not limited to,phenylalanine (F) or tryptophan (W). Also provided are modified FIXpolypeptides that contain an amino acid replacement at residue R403 orat a residue in a FIX polypeptide corresponding to 403 that is aglutamic acid, e.g., R403E, or is a conservative amino acid replacementthereof. For example, conservative amino acid residues for glutamic acidinclude, but are not limited to, aspartic acid (D).

In a further embodiment, combination mutants can be generated. Includedamong such combination mutants are those having two or more mutations atamino acid positions 202, 203, 204, 257, 239, 293, 312, 316, 318, 333,338, 400, 403 and 410 (corresponding to amino acid positions 38, 39, 40,74, 92, 126, 143, 148, 150, 165, 170, 230, 233 and 240, respectively, bychymotrypsin numbering). For example, a modified FIX polypeptide canpossess amino acid substitutions at 2, 3, 4, 5 or more of the identifiedpositions. Hence, a modified polypeptide can display 1, 2, 3, 4, 5 ormore mutations that can result in increased resistance of the modifiedFIX polypeptide to the inhibitory effects of AT-III, AT-III/heparinand/or heparin. Any one or more of the mutations described herein toincrease resistance of the modified FIX polypeptide to the inhibitoryeffects of AT-III, AT-III/heparin and/or heparin can be combined.

Table 6 provides non-limiting examples of exemplary amino acidreplacements at the identified residues, corresponding to amino acidpositions of a mature FIX polypeptide as set forth in SEQ ID NO:3.Included amongst these are exemplary combination mutations. As noted,such FIX polypeptides are designed to increase resistance to AT-III,AT-III/heparin and/or heparin, and therefore have increased coagulantactivity in vivo, ex vivo, or in in vitro assays that include ATIII,heparin/ATIII, heparin, plasma, serum, or blood. In reference to suchmutations, the first amino acid (one-letter abbreviation) corresponds tothe amino acid that is replaced, the number corresponds to the positionin the mature FIX polypeptide sequence with reference to SEQ ID NO:3,and the second amino acid (one-letter abbreviation) corresponds to theamino acid selected that replaces the first amino acid at that position.The amino acid positions for mutation also are referred to by thechymotrypsin numbering scheme. In Table 6 below, the sequence identifier(SEQ ID NO) is identified in which exemplary amino acid sequences of themodified FIX polypeptide are set forth.

TABLE 6 Mutation Mutation SEQ ID (Mature FIX Numbering) (ChymotrypsinNumbering) NO R318A R150A 120 R318E R150E 121 R318Y R150Y 122 R318FR150F 413 R318W R150W 414 R312Q R143Q 123 R312A R143A 124 R312Y R143Y125 R312L R143L 126 V202M V38M 127 V202Y V38Y 128 D203M D39M 129 D203YD39Y 130 A204M A40M 131 A204Y A40Y 132 K400A/R403A K230A/R233A 133K400E/R403E K230E/R233E 134 R403A R233A 135 R403E R233E 136 R403D R233D417 K400A K230A 137 K400E K230E 138 K293E K126E 139 K293A K126A 140R333A R165A 141 R333E R165E 142 R333S R165S 186 R338A R170A 143 R338ER170E 144 R338L R170L 187 R338A/R403A R170A/R233A 145 R338E/R403ER170E/R233E 146 K293A/R403A K126A/R233A 147 K293E/R403E K126E/R233E 148K293A/R338A/R403A K126A/R170A/R233A 149 K293E/R338E/R403EK126E/R170E/R233E 150 R318A/R403A R150A/R233A 151 R318E/R403ER150E/R233E 152 R318Y/R338E/R403E R150Y/R170E/R233E 156 R318Y/R338ER150Y/R170E 188 R318N/A320S R150N/A152S 119 K316N K148N 189 K316A K148A190 K316E K148E 191 K316S K148S 192 K316M K148M 193 E239N E74N 109 E239SE74S 194 E239A E74A 195 E239R E74R 196 E239K E74K 197 H257F H92F 198H257Y H92Y 199 H257E H92E 200 H257S H92S 201 E410N E240N 108 N260S N95S116 F314N/K316S F145N/K148S 113

The modifications described herein to increase resistance to aninhibitor, such as AT-III and/or heparin, can be combined with any othermutation described herein or known in the art. Typically, the resultingmodified FIX polypeptide exhibits increased coagulant activity comparedto an unmodified FIX polypeptide. For example, one or more modificationsthat increase resistance to an inhibitor, such as AT-III and/or heparin,can be combined with modification(s) that introduce a non-nativeglycosylation site, eliminate one or more native glycosylation sites,eliminate one or more of the native sulfation, phosphorylation orhydroxylation sites, increase catalytic activity, increase intrinsicactivity, increase binding to phospholipids, or improve pharmacokineticand/or pharmacodynamic properties. The resulting modified FIXpolypeptide typically exhibits increased coagulant activity compared toan unmodified FIX polypeptide.

Modified FIX polypeptides that have increased resistance for AT-IIIalone, the AT-III/heparin complex and/or heparin alone, can exhibit areduction in the affinity for heparin, the extent of inhibition underspecified conditions, or in the second order rate constant forinhibition by ATIII or heparin/ATIII at least or at least about 1%, 2%,3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,95%, 99% or more compared to the affinity, extent of inhibition, or thesecond order rate constant for inhibition of unmodified or wild-type FIXpolypeptide either in vivo or in vitro. Thus, the modified FIXpolypeptides can exhibit increased resistance to AT-III alone, theAT-III/heparin complex and/or heparin alone that is at least or at leastabout 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more of the resistanceexhibited by an unmodified FIX polypeptide. Increased resistance toAT-III, the AT-III/heparin complex and/or heparin by such modified FIXpolypeptides also can be manifested as increased coagulation activity orimproved duration of coagulation activity in vivo or in vitro in thepresence of AT-III, the AT-III/heparin complex, heparin, blood, plasma,or serum. The coagulation activity of the modified FIX polypeptides canbe increased by at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, ormore compared to the coagulation activity of unmodified or wild-type FIXpolypeptide either in vivo or in vitro. Modified FIX polypeptidescontaining modifications that increase resistance to AT-III, theheparin/AT-III complex, and/or heparin also can exhibit an enhancedtherapeutic index compared with unmodified FIXa.

c. Mutations to Increase Catalytic Activity

The modified FIX polypeptides provided herein can contain one or moremodifications to increase the catalytic activity of the polypeptidecompared to an unmodified FIX. For example, modifications can be made tothe amino acids that are involved in the interaction of FIX with itscofactor, FVIIIa, such that the resulting modified FIX polypeptide hasincreased affinity for FVIIIa, and thereby displays increased activitytoward FX under conditions in which FVIIIa is not present at saturatingconcentrations. Modifications also can be made to the protease domain ofthe FIX polypeptide, such that the activity or catalytic efficiency ofthe modified FIX polypeptide for activation of FX, in the presenceand/or absence of the co-factor FVIIIa, is increased compared to theactivity or catalytic efficiency of the unmodified polypeptide.

Exemplary modifications that can be included in the modified FIXpolypeptides provided herein include amino acid replacements atpositions 259, 265, 345, 410 and 412 (corresponding to 94, 98, 177, 240and 242 by chymotrypsin numbering). The amino acids at these positionscan be replaced by any other amino acid residue. In some examples, thetyrosine at position 259 is replaced with a phenylalanine; the lysine atposition 265 is replaced with a threonine; and/or the tyrosine atposition 345 is replaced with a threonine. In further example, theglutamic acid at position 410 is replaced with a glutamine, serine,alanine or aspartic acid. In one example, the threonine at position 412is replaced with a valine or an alanine.

The above mentioned modifications are exemplary only. Many othermodifications described herein also result in increased catalyticactivity. For example, modifications that are introduced into the FIXpolypeptide to increase resistance to an inhibitor, such as AT-IIIand/or heparin, introduce a non-native glycosylation site, eliminate oneor more native glycosylation sites, eliminate one or more of the nativesulfation, phosphorylation or hydroxylation sites, increase intrinsicactivity, increase binding to phospholipids, decrease binding to LRP,and/or improve pharmacokinetic and/or pharmacodynamic properties, canalso result in a modified FIX polypeptide that exhibits increasedactivity.

Table 7 provides non-limiting examples of exemplary amino acidreplacements at the identified residues, corresponding to amino acidpositions of a mature FIX polypeptide as set forth in SEQ ID NO:3. Inreference to such mutations, the first amino acid (one-letterabbreviation) corresponds to the amino acid that is replaced, the numbercorresponds to the position in the mature FIX polypeptide sequence withreference to SEQ ID NO:3, and the second amino acid (one-letterabbreviation) corresponds to the amino acid selected that replaces thefirst amino acid at that position. The amino acid positions for mutationalso are referred to by the chymotrypsin numbering scheme. In Table 7below, the sequence identifier (SEQ ID NO) is identified in whichexemplary amino acid sequences of the modified FIX polypeptide are setforth.

TABLE 7 Mutation Mutation SEQ ID (Mature FIX Numbering) (ChymotrypsinNumbering) NO T412A T242A 202 T412V T242V 203 E410Q E240Q 174 E410SE240S 175 E410A E240A 176 E410D E240D 206 Y259F/K265T/Y345TY94F/K98T/Y177T 216

The modifications described herein to increase catalytic activity can becombined with any other mutation described herein or known in the art.Typically, the resulting modified FIX polypeptide exhibits increasedcoagulant activity compared to an unmodified FIX polypeptide. Forexample, one or more modifications that increase catalytic activity canbe combined with modification(s) that increase resistance to aninhibitor, such as AT-III and/or heparin, introduce a non-nativeglycosylation site, eliminate one or more native glycosylation sites,eliminate one or more of the native sulfation, phosphorylation orhydroxylation sites, increase intrinsic activity, increase binding tophospholipids, or improve pharmacokinetic and/or pharmacodynamicproperties. The resulting modified FIX polypeptide typically exhibitsincreased coagulant activity compared to an unmodified FIX polypeptide.

Modified FIX polypeptides that have increased catalytic activity canexhibit at least or about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more activity compared tothe catalytic activity of unmodified or wild-type FIX polypeptide eitherin vivo or in vitro. Increased catalytic activity of such modified FIXpolypeptides also can be manifested as increased coagulation activity,duration of coagulation activity and/or enhanced therapeutic index. Thecoagulation activity of the modified FIX polypeptides can be increasedby at least or at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, ormore compared to the coagulation activity of unmodified or wild-type FIXpolypeptide either in vivo or in vitro.

d. Mutations to Decrease LRP Binding

FIXa can be cleared from systemic circulation by binding the low-densitylipoprotein receptor-related protein (LRP), which is a membraneglycoprotein that is expressed on a variety of tissues, including liver,brain, placenta and lung. Thus, provided herein are modified FIXpolypeptides that exhibit decreased binding to the LRP. This can resultin improved pharmacokinetic properties of the modified FIX polypeptide,including, for example, i) decreased clearance, ii) altered volume ofdistribution, iii) enhanced in vivo recovery, iv) enhanced total proteinexposure in vivo (i.e., AUC), v) increased serum half-life (α, β, and/orγ phase), and/or vi) increased mean resonance time (MRT). Such modifiedFIX polypeptides can exhibit increased coagulant activity.

The modified FIX polypeptide provided herein can contain one or moremodifications in the LRP-binding site. This binding site is postulatedto be located in a loop in the protease domain spanning residues 342 to346 of the mature FIX polypeptide set forth in SEQ ID NO:3. Modificationof one or more of the residues at positions 342-346 (corresponding topositions 174-178 by chymotrypsin numbering), such as by amino acidreplacement, insertion or deletion, can interfere with the interactionbetween the modified FIX polypeptide and LRP, resulting in decreasedbinding affinity. The binding of the modified FIX polypeptides to LRPcan be tested using assays known to one of skill in the art (see, e.g.Rohlena et al., (2003) J. Biol. Chem. 278:9394-9401). The resultingimproved pharmacokinetic properties also can be tested using well knownin vivo assays, including those described below.

Exemplary modifications that can be included in the modified FIXpolypeptides provided herein include amino acid replacements atpositions 343, 344, 345 and 346 (corresponding to 175, 176, 177 and 178by chymotrypsin numbering). The amino acids at these positions can bereplaced by any other amino acid residue. In some examples, thethreonine at position 343 is replaced with a glutamine, glutamic acid,aspartic acid or arginine; the phenylalanine at position 344 is replacedwith an isoleucine; the tyrosine at position 345 is replaced with athreonine, alanine or an alanine; and/or the asparagine at position 346is replaced with an aspartic acid or a tyrosine. Any one or more ofthese exemplary amino acid replacements can be combined with each otheror with other modifications described herein.

Provided herein are modified FIX polypeptides that contain an amino acidreplacement at residue T343 or at a residue in a FIX polypeptidecorresponding to 343 that is an arginine, e.g., T343R, or is aconservative amino acid replacement thereof. For example, conservativeamino acid residues for arginine include, but are not limited to, lysine(K).

Table 8 provides non-limiting examples of exemplary amino acidreplacements at the identified residues, corresponding to amino acidpositions of a mature FIX polypeptide as set forth in SEQ ID NO:3. Inreference to such mutations, the first amino acid (one-letterabbreviation) corresponds to the amino acid that is replaced, the numbercorresponds to the position in the mature FIX polypeptide sequence withreference to SEQ ID NO:3, and the second amino acid (one-letterabbreviation) corresponds to the amino acid selected that replaces thefirst amino acid at that position. The amino acid positions for mutationalso are referred to by the chymotrypsin numbering scheme. In Table 8below, the sequence identifier (SEQ ID NO) is identified in whichexemplary amino acid sequences of the modified FIX polypeptide are setforth.

TABLE 8 Mutation Mutation SEQ ID (Mature FIX Numbering) (ChymotrypsinNumbering) NO N346D N178D 207 N346Y N178Y 208 T343R T175R 209 T343ET175E 210 T343D T175D 416 T343Q T175Q 211 F342I F174I 212 Y345A Y177A213 Y345T Y177T 214 T343R/Y345T T175R/Y177T 215 T343R/N346D T175R/N178D409 T343R/N346Y T175R/N178Y 410

The modifications described herein to decrease binding to LRP can becombined with any other mutation described herein or known in the art.Typically, the resulting modified FIX polypeptide exhibits increasedcoagulant activity compared to an unmodified FIX polypeptide. Forexample, one or more modifications that decrease binding to LRP can becombined with modification(s) that increase resistance to an inhibitor,such as AT-III and/or heparin, increase catalytic activity, introduce anon-native glycosylation site, eliminate one or more nativeglycosylation sites, eliminate one or more of the native sulfation,phosphorylation or hydroxylation sites, increase activity in thepresence and/or absence of FVIIIa, increase binding to phospholipids, orimprove pharmacokinetic and/or pharmacodynamic properties. The resultingmodified FIX polypeptide typically exhibits increased coagulant activitycompared to an unmodified FIX polypeptide.

Modified FIX polypeptides that have decreased binding to LRP can exhibitat a decrease of at least or about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more comparedto the binding of unmodified or wild-type FIX polypeptide to LRP invitro. Decreased binding to LRP by such modified FIX polypeptides canresult in improved pharmacokinetic properties, such as i) decreasedclearance, ii) altered volume of distribution, iii) enhanced in vivorecovery, iv) enhanced total protein exposure in vivo (i.e., AUC), v)increased serum half-life (αγ, β, and/or γ phase), and/or vi) increasedmean resonance time (MRT). Further, such alterations can result inincreased coagulant activity, duration of coagulation activity and/orenhanced therapeutic index. The coagulation activity of the modified FIXpolypeptides can be increased by at least or at least about 1%, 2%, 3%,4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,100%, 200%, 300%, 400%, 500%, or more compared to the coagulationactivity of unmodified or wild-type FIX polypeptide either in vivo or invitro.

e. Other Mutations to Alter Posttranslational Modification

Wild-type FIX is posttranslationally modified upon expression inmammalian cells. The Factor IX precursor polypeptide undergoes extensiveposttranslational modification to become the mature zymogen that issecreted into the blood. Such posttranslational modifications includeγ-carboxylation, β-hydroxylation, O- and N-linked glycosylation,sulfation and phosphorylation. As discussed above, the levels ofglycosylation can be altered by, for example, introducing new non-nativeglycosylation sites and/or eliminating native glycosylation sites.Similarly, other posttranslational modifications can be altered, such asby introducing and/or eliminating γ-carboxylation, β-hydroxylation,sulfation and/or phosphorylation sites.

Any one or more of the native γ-carboxylation, β-hydroxylation,sulfation or phosphorylation sites can be eliminated, such as by aminoacid replacement or deletion. For example, unmodified FIX polypeptidescan be modified by amino acid replacement of any one or more of thetwelve glutamic acid residues (corresponding to positions 7, 8, 15, 17,20, 21, 26, 27, 30, 33, 36 and 40 of the mature FIX set forth in SEQ IDNO:3) in the Gla domain. These residues typically are γ-carboxylated toγ-carboxyglutamyl (or Gla) in wild-type FIX. Thus, removal of theglutamic acid residues, such as by amino acid substitution or deletion,can reduce the level of γ-carboxylation in a modified FIX polypeptidecompared to the unmodified FIX polypeptide. Similarly, the aspartic acidresidue at position 64, which normally is β-hydroxylated in wild-typeFIX, can be removed, such as by amino acid substitution or deletion.Additional post-translational modification sites that can be eliminatedinclude, for example, the tyrosine at position 155, which typically issulfated in wild-type FIX, and the serine residue at position 158, whichtypically is phosphorylated in wild-type FIX.

In other examples, non-native post-translational modification sites canbe introduced, such as by amino acid replacement or insertion. Forexample, additional glutamic acid residues can be introduced into theGla domain. Such glutamic acid residues could be γ-carboxylated toγ-carboxyglutamyl (or Gla) in the modified FIX polypeptide uponexpression in, for example, a mammalian cell. Similarly, one or morenon-native β-hydroxylation, sulfation or phosphorylation sites can beintroduced.

Provided herein are modified FIX polypeptides that have one or more ofthe native posttranslational modification sites eliminated. The modifiedFIX polypeptides that have been modified to eliminate one or morepost-translational modification sites, including γ-carboxylation,β-hydroxylation, sulfation and/or phosphorylation sites, retain at leastone activity of the unmodified FIX polypeptide. In some examples, themodified FIX polypeptide retains at least or at least about 5%, 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more of the catalyticactivity of the unmodified FIX polypeptide. In other examples, themodified FIX polypeptide retains at least or at lest about 5%, 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more of the binding activityfor FVIIIa of the unmodified FIX polypeptide. In some assays and/orunder some conditions, the modified FIX polypeptides can exhibitincreased activity compared with the unmodified FIX protein (e.g.,increased pharmacodynamic activity in vivo, and/or activity in thepresence of AT-III/heparin or plasma).

Provided herein are modified FIX polypeptides that contains an aminoacid replacement at residue Y155 or at a residue in a FIX polypeptidecorresponding to 155 that is a phenylalanine, e.g., Y155F, or is aconservative amino acid replacement thereof. For example, conservativeamino acid residues for phenylalanine include, but are not limited to,methionine (M), leucine (L) or tyrosine (Y).

Table 9 provides non-limiting examples of exemplary amino acidreplacements, corresponding to amino acid positions of a mature FIXpolypeptide as set forth in SEQ ID NO:3, that are included in a modifiedFIX polypeptide to eliminate a native β-hydroxylation, sulfation and/orphosphorylation sites at positions 64, 155 and 158, respectively. InTable 9 below, the sequence identifier (SEQ ID NO) is identified inwhich exemplary amino acid sequences of the modified FIX polypeptide areset forth.

TABLE 9 Mutation Mutation SEQ ID (Mature FIX Numbering) (ChymotrypsinNumbering) NO D64N D[64]N 83 D64A D[64]A 84 Y155F Y[155]F 76 Y155HY[155]H 93 Y155Q Y[155]Q 94 T155L Y[155]L 415 S158A S[158]A 95 S158DS[158]D 96 S158E S[158]E 97

The modifications described herein to eliminate β-hydroxylation,sulfation and/or phosphorylation sites can be combined with any othermutation described herein or known in the art. Typically, the resultingmodified FIX polypeptide exhibits increased coagulant activity comparedto an unmodified FIX polypeptide. For example, one or more modificationsthat eliminate one or more native β-hydroxylation, sulfation and/orphosphorylation sites can be combined with modification(s) that increaseresistance to an inhibitor, such as AT-III and/or heparin, alterglycosylation, such as increase glycosylation, increase catalyticactivity, increase intrinsic activity, increase binding tophospholipids, or improve pharmacokinetic and/or pharmacodynamicproperties.

The modified FIX polypeptides provided herein that eliminate one or morenative β-hydroxylation, sulfation and/or phosphorylation sites retain atleast one activity of FIX, such as, for example, catalytic activity forits substrate, FX, or binding to the co-factor, FVIIIa. Typically, themodified FIX polypeptides provided herein retain at least or at leastabout 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more ofthe catalytic activity exhibited by an unmodified FIX polypeptide. Insome instances, the coagulant activity of the modified FIX polypeptidesis increased by at least or at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%,400%, 500%, or more compared to the coagulation activity of unmodifiedor wild-type FIX polypeptide either in vivo or in vitro.

2. Combination Modifications

The modified FIX polypeptides provided herein that contain one or morenon-native glycosylation sites, have one or more native glycosylationsites eliminated, have one or more native β-hydroxylation, sulfationand/or phosphorylation sites eliminated, or that have modifications thatcan result in increased resistance to inhibitors, such as AT-III,AT-III/heparin and/or heparin, compared to a wild-type FIX polypeptide,also can contain other modifications. In some examples, the modified FIXpolypeptides contain modifications that introduce one or more non-nativeglycosylation sites and also contain modifications that interfere withthe interaction between FIX and inhibitors, such as AT-III, theAT-III/heparin complex and/or and heparin. In other examples,modifications that eliminate one or more native β-hydroxylation,sulfation and/or phosphorylation sites can be combined withmodifications that increase resistance to inhibitors, and/ormodifications that introduce one or more glycosylation sites. Thus, oneor more of the mutations set forth in Tables 3-9 above, can be combinedwith any of the other mutations set forth in Tables 3-9 above. Thus,included among the modified FIX polypeptides provided herein are thosethat exhibit increased glycosylation, such as N-glycosylation; increasedresistance to AT-III, AT-III/heparin, and/or heparin; decreasedβ-hydroxylation, sulfation and/or phosphorylation; and/or increasedcatalytic activity compared with an unmodified FIX polypeptide.

Further, any of the modified FIX polypeptides provided herein cancontain any one or more additional modifications. In some examples, theadditional modifications result in altered properties and/or activitiescompared to an unmodified FIX polypeptide. Typically, such additionalmodifications are those that themselves result in an increased coagulantactivity of the modified polypeptide and/or increased stability of thepolypeptide. Accordingly, the resulting modified FIX polypeptidestypically exhibit increased coagulant activity.

The additional modifications can include, for example, any amino acidsubstitution, deletion or insertion known in the art, typically any thatincreases the coagulant activity and/or stability of the FIXpolypeptide. Any modified FIX polypeptide provided herein can contain 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 ormore additional amino acid modifications. Typically, the resultingmodified FIX polypeptide retains at least one activity of the wild-typeor unmodified polypeptide, such as, for example, catalytic activity, orbinding to the co-factor, FVIIIa.

Additional modifications in the primary sequence can be made to the FIXpolypeptide to effect post-translational modifications. For example, themodified FIX polypeptides provided herein can contain non-nativeglycosylation sites including and other than those described above, suchas any of those described in the art, including non-native O-linked orS-linked glycosylation sites described in U.S. Patent Publication No.20080280818, or the non-native glycosylation sites described inInternational Patent Publication Nos. WO20091300198 and WO2009137254.

In other examples, the additional modification can be made to the FIXpolypeptide sequence such that its interaction with other factors,molecules and proteins is altered. For example, the amino acid residuesthat are involved in the interaction with Factor X can be modified suchthat the affinity and/or binding of the modified FIX polypeptide to FXis increased. Other modifications include, but are not limited to,modification of amino acids that are involved in interactions withFVIIIa, heparin, antithrombin III and phospholipids.

Additional modifications also can be made to a modified FIX polypeptideprovided herein that alter the conformation or folding of thepolypeptide. These include, for example, the replacement of one or moreamino acids with a cysteine such that a new disulphide bond is formed,or modifications that stabilize an α-helix conformation, therebyimparting increased activity to the modified FIX polypeptide.

Modifications also can be made to introduce amino acid residues that canbe subsequently linked to a moiety, such as one that acts to increasestability of the modified FIX polypeptide. For example, cysteineresidues can be introduced to facilitate conjugation to a polymer, suchpolyethylene glycol (PEG) (International Pat. Pub. No. WO2009140015).The stability of a FIX polypeptide also can be altered by modifyingpotential proteolytic sites, such as removing potential proteolyticsites, thereby increasing the resistance of the modified FIX polypeptideto proteases (see e.g. US Pat. Pub. No. 20080102115).

Additionally, amino acids substitutions, deletions or insertions can bemade in the endogenous Gla domain such that the modified FIX polypeptidedisplays increased binding and/or affinity for phospholipid membranes.Such modifications can include single amino acid substitution, deletionsand/or insertions, or can include amino acid substitution, deletion orinsertion of multiple amino acids. For example, all or part of theendogenous Gla domain can be replaced with all or part of a heterologousGla domain. In other examples, the modified FIX polypeptides providedherein can display deletions in the endogenous Gla domain, orsubstitutions in the positions that are normally gamma-carboxylated.Alternatively, amino acid substitutions can be made to introduceadditional, potential gamma-carboxylation sites.

The following sections describe non-limiting examples of exemplarymodifications described in the art to effect increased stability and/orcoagulant activity of a FIX polypeptide. As discussed above, suchmodifications also can be additionally included in any modified FIXpolypeptide provided herein. The amino acid positions referenced belowcorrespond to the mature FIX polypeptide as set forth in SEQ ID NO:3.Corresponding mutations can be made in other FIX polypeptides, such asallelic, species or splice variants of the mature FIX polypeptide setforth in SEQ ID NO:3.

a. Modifications to Increase Activity

In one example, additional modifications can be made to a modifiedfactor IX polypeptide provided herein that result in increased catalyticactivity toward factor X. For example, modifications can be made to theamino acids that are involved in the interaction with its cofactor,FVIIIa, such that the resulting modified FIX polypeptide has increasedaffinity for FVIIIa, and thereby displays increased activity toward FXunder conditions in which FVIIIa is not saturating. Modifications canalso be made in FIX that increase the catalytic efficiency of FIXapolypeptides and/or the FIXa/FVIIIa complex, compared to the activity ofthe unmodified FIXa polypeptide or FIXa/FVIIIa complex, for activationof the substrate FX.

Examples of additional modifications that can be included in themodified FIX polypeptides provided herein to increase the intrinsicactivity of the modified FIX polypeptide include, but are not limitedto, those described in Hopfner et al., (1997) EMBO J. 16:6626-6635;Kolkman et al., (2000) Biochem. 39:7398-7405; Sichler et al., (2003) J.Biol. Chem. 278:4121-4126; Begbie et al., (2005) Thromb. Haemost.94(6):1138-47; U.S. Pat. No. 6,531,298 and U.S. Patent Publication Nos.20080167219 and 20080214461. Non-limiting examples of exemplary aminoacid modifications described in the art that can result in increasedintrinsic activity of the modified FIX polypeptide include any one ormore of V86A, V86N, V86D, V86E, V86Q, V86G, V86H, V86I, V86L, V86M,V86F, V86S, V86T, V86W, V86Y, Y259F, A261K, K265T, E277V, E277A, E277N,E277D, E277Q, E277G, E277H, E2771, E277L, E277M, E277F, E277S, E277T,E277W, E277Y, R338A, R338V, R3381, R338F, R338W, R338S, R338T, Y345F,I383V, E388G. For example, a modified FIX polypeptide provided hereincan contain the amino acid substitutions Y259F/K265T, Y259F/K265T/Y345F,Y259F/A261K/K265T/Y345F, Y259F/K265T/Y345F/I383V/E388G orY259F/A261K/K265T/Y345F/I383V/E388G. In another example, the modifiedFIX polypeptides provided herein can contain modifications that removethe activation peptide (Δ155-177) (see, e.g. Begbie et al., (2005)Thromb. Haemost. 94(6):1138-47), which can both increase activity anddecrease clearance in vivo.

b. Modifications that Increase Affinity for Phospholipids or ReduceBinding to Collagen

The modified FIX polypeptides provided herein also can contain one ormore additional modifications to increase affinity for phospholipids.The coagulant activity of FIX can be enhanced by increasing the bindingand/or affinity of the polypeptide for phospholipids, such as thoseexpressed on the surface of activated platelets. This can be achieved,for example, by modifying the endogenous FIX Gla domain. Modificationcan be effected by amino acid substitution at one or more positions inthe Gla domain of a FIX polypeptide that result in a modified FIXpolypeptide with increased ability to bind phosphatidylserine and othernegatively charged phospholipids. Examples of additional modificationsto increase phospholipid binding and/or affinity and that can be made toa modified FIX polypeptide provided herein, include, but are not limitedto, those described in U.S. Pat. No. 6,017,882. For example, a modifiedFIX polypeptide provided herein can contain one or more modifications atamino acid positions 11, 12, 29, 33 and/or 34 (corresponding to a matureFIX polypeptide set forth in SEQ ID NO:3). Exemplary of suchmodifications are amino acid substitutions K51, K5L, K5F, K5E, Q11E,Q11D, R16E, R29F and/or N34E, N34D, N34F, N341, N34L, T35D and T35E.

In another aspect, the modified FIX polypeptides provided herein alsocan contain one or more additional modifications to reduce affinity forcollagen. The coagulant activity of FIX can be enhanced by reducing thebinding and/or affinity of the polypeptide for collagen IV, which ispresent on the surface of the extracellular matrix on endothelial cells.A reduced binding to collagen IV can result in increased circulation ofthe modified FIX polypeptides and, thus, increased coagulant activity invivo. This can be achieved, for example, by modifying the FIX Gla domainat amino acid residues 3 to 11 of a mature FIX polypeptide set forth inSEQ ID NO:3, which are responsible for the interaction with collagen IV(Cheung et al., (1992) J. Biol. Chem. 267:20529-20531; Cheung et al.,(1996) Proc. Natl. Acad. Sci. U.S.A. 93:11068-11073). Modification canbe effected by amino acid substitution at one or more positions in theGla domain of a FIX polypeptide that result in a modified FIXpolypeptide with decreased ability to bind collagen IV. Examples ofadditional modifications to increase phospholipid binding and/oraffinity and that can be made to a modified FIX polypeptide providedherein, include, but are not limited to, those described in Schuettrumpfet al., (2005) Blood 105(6):2316-23; Melton et al., (2001) Blood Coagul.Fibrinolysis 12(4):237-43; and Cheung et al., (1996) Proc. Natl. Acad.Sci. U.S.A. 93:11068-11073. For example, a modified FIX polypeptideprovided herein can contain are amino acid substitutions K5A and/orV10K.

c. Additional Modifications to Increase Resistance to Inhibitors

Additional modifications can be included that increase the activity ofthe FIX polypeptide by increasing the resistance of the modified FIXpolypeptide to inhibitors, such as, for example, inhibition byantithrombin III (AT-III)/heparin. Typically, this can be achieved bymodifying one or more residues that are involved in the interaction withAT-III, heparin or the AT-III/heparin complex. Exemplary of suchmodifications include those described, for example, in U.S. Pat. No.7,125,841; U.S. Pat. Pub. No 20040110675; Int. Pat. Pub. No.WO2002040544; Chang, J. et al., (1998) J. Biol. Chem. 273(20):12089-94;Yang, L. et al., (2002) J. Biol. Chem. 277(52):50756-60; Yang, L. etal., (2003) J. Biol. Chem. 278(27):25032-8; Rohlena et al., (2003) J.Biol. Chem. 278(11):9394-401; Sheehan et al., (2006) Blood107(10):3876-82; Buyue et al. (2008) Blood 112:3234-3241. Non-limitingexamples of modifications that can be included to decrease inhibition byAT-III and/or heparin, include, but are not limited to, modifications atamino acid positions corresponding to amino acid positions R252, H256,H257, K265, H268, K293, R318, R333, R338, K400, R403, K409 or K411 of amature FIX polypeptide set forth in SEQ ID NO:3. For example, the FIXpolypeptides provided herein can contain the amino acid substitutionsR252A, H257A, H268A, K293A, R318A, R333A, R338A, K400A, R403A, R403Eand/or K411A.

d. Additional Modifications to Alter Glycosylation

Modifications, in addition to those described above can be incorporatedinto the modified FIX polypeptides provided herein to alter theglycosylation of the modified FIX polypeptides compared to an unmodifiedFIX polypeptide. For example, the modified FIX polypeptides can containone or more modifications that introduce one or more non-nativeglycosylation sites into the modified FIX polypeptide. Thus, whenexpressed in an appropriate system, the modified FIX polypeptides canexhibit altered glycosylation patterns compared to an unmodified FIXpolypeptide. In some examples, the modified FIX polypeptides exhibitincreased glycosylation compared to an unmodified FIX polypeptide, suchas increased N-glycosylation or increased O-glycosylation

Examples of additional modifications that can be included in themodified FIX polypeptides provided herein to alter the glycosylationprofile of a FIX polypeptide include, but are not limited to, thosedescribed in International Published Application Nos. WO2009130198,WO2009051717 and WO2009137254. Exemplary modifications that can beincluded in a modified FIX polypeptide provided herein to increaseglycosylation include, but are not limited to, Y1N, Y1N+S3T, S3N+K5S/T,G4T, G4N+L6S/T, K5N+E7T, L6N+E8T, E7N+F9T, F9N+Q11S/T, V10N+G12S/T,Q11N+N13T, G12N+L14S/T, L14N+R16T, E15T, E15N+E17T; R16N+C18S/T,M19N+E21T; E20N+K22T, K22N, S24N+E26T; F25N+E27T; E26N+A28T; E27N+R29T;A28N+E30T; R29N+V31S/T, E30N+F32T; V31N+E33T; F32N+N34T, E33N,T35N+R37S/T, E36T; E36N; R37N, T39N+F41S/T, E40N+W42T, F41N+K43S/T,W42N+Q44S/T, K43N+Y45T; Q44N+V46S/T, Y45N+D47T, V46N+G48S/T,D47N+D49S/T, G48N+Q50S/T, D49N+C51S/T, Q50N+E52S/T, E52N+N54T,S53N+P55S/T, C56S/T, L57N+G59S/T, G59N+S61T; G60S/T, S61N+K63S/T,K63N+D65S/T, D65N+N67S/T, I66N+S68S/T, Y69S/T, Y69N+C71S/T, S68N+E70S/T,E70N+W72S/T, W72N+P74S/T, P74N+G76S/T, F75N, G76N+E78T, E78N+K80T, F77T,F77N+G79S/T, G79N+N81S/T, K80N+C82S/T, E83S/T, E83N+D85S/T, L84N+V86S/T,D85N, V86A, V86N+C88S/T, T87N+N89S/T, 190N+N92S/T, K91S/T, 190N+N92S/T,K91N+G93S/T, R94S/T, R94N+E96S/T, K100N, A103S/T, S102N+D104S/T,A103N+N105S/T, D104N+K106S/T, V107S/T, K106N+V108S/T, V108N+V110S/T,S111N, E113N+Y115S/T, G114N+R116S/T, R116N+A118S/T, E119N+Q121S/T,K122S/T, Q121N+S123S/T, K122N+C124S/T S123N+E125S/T, E125N+A125S/T,P126N+V128S/T, A127N+P129T, V128N+F130S/T, P129N+P131S/T, F130N+C132S/T,R134N, V135N+V137S/T, S136N, S138N, V137N+Q139T; Q139N, T140N+L142S/T,S141N+L143S/T, K142N, A146N+A148S/T, E147N+V149S/T, T148N+F150S/T,V149N+P151S/T, F150N+D152S/T, P151N+V153S/T, D152N+D154S/T,V153N+Y155S/T, D154N+V156S/T, Y155N+N157S/T, V156N, S158N+E160S/T,T159N+A161S/T, E160N+E162S/T, A161N, E162N+1164S/T, T163N+L165S/T,I164N+D166S/T, L165N+N167S/T, D166N+1168S/T, 1168N+Q170S/T, T169N,Q170N, S171N+Q173S/T, T172N, Q173N+F175S/T, S174N+N176S/T,F175N+D177S/T, F178S/T, D177N, D177E, F178N+R180S/T, T179N+V181S/T,R180N+V182S/T, G183+E185S/T, G184N+D186T, E185N+A187S/T, D186N+K188S/T,A187N+P189T, K188N+G190S/T, P189N+Q181S/T, G200N+V202T, K201N+D203S/T,K201T, V202N+A204S/T, D203N+F205S/T, E213N+W215S/T, K214T, V223T,E224N+G226S/T, T225N+V227S/T, G226N+K228S/T, V227N+1229T, K228N,H236N+1238T; 1238N+E240T; E239N, E240N+E242S/T, E242N, T241N+H243S/T,H243N+E245S/T, K247N+N249S/T, V250N+R252T, I251S/T, I251N+12535/T,R252N+12545/T, 1253N+P255S/T, P255N+H257S/T, H257N+Y259S/T, N260S/T,A262S/T, A261N+1263 S/T, A262N+N264S/T, I263N+K265S/T, K265N+N267S/T,A266N+H268S/T, D276N+P278S/T, P278N+V280S/T, E277N+L279S/T,V280N+N282S/T, Y284S/T, S283N+V285S/T, Y284N, D292N+K294S/T,K293N+Y295S/T, E294N, F299S/T, I298N+L300S/T, K301N+G303S/T, F302N,G303N+G305S/T, S304N+Y306S/T, Y306N+S308S/T, R312N+F314S/T, V313N+H315T,F314N+K316S/T, H315N+G317S/T, K316N+R138S/T, G317N, R318N+A320S/T,S319N+L321S/T, A320N+V322T, L321N+L323S/T, V322N+Q324S/T, Y325N+R327S/T,R327N+P329S/T, P329N+V331S/T, L330N+D332S/T, D332N+A334S/T, R333N,A334N+C336S/T, T335N+L337S/T, L337N, R338N, S339N+K341T, T340N+F342T;K341N, F342N+I344S/T, T343N+Y345S/T, Y345N+N347S/T, M348S/T,G352N+H354T, F353N, F353N+E355T, H354N+G356S/T, H354V, H3541, E355T,E355N+G357S/T, G356N+R358T, G357N+D359S/T, R358N, Q362N+D364S/T, V370N;T371V; T3711; E372T, E372N+E374S/T, E374N, G375N, W385N+E387T;G386N+E388T, E388N+A390S/T, A390N+K392T, M391N+G393S/T, K392N+K394S/T,K392V, G393T, G393N+Y395S/T, K394N+G396S/T, R403N+V405S/T, 1408S/T,K409N+K411S/T, E410N, K411N+K413S/T, and K413N.

e. Modifications to Increase Resistance to Proteases

Modified FIX polypeptides provided herein also can contain additionalmodifications that result in increased resistance of the polypeptide toproteases. For example, amino acid substitutions can be made that removeone or more potential proteolytic cleavage sites. The modified FIXpolypeptides can thus be made more resistant to proteases, therebyincreasing the stability and half-life of the modified polypeptide.

Examples of additional modifications that can be included in themodified FIX polypeptides provided herein to increase resistance toproteases include, but are not limited to, those described in U.S.Patent Publication No. 20080102115 and International PublishedApplication No. WO2007149406. Exemplary modifications that can beincluded in a modified FIX polypeptide provided herein to increaseprotease resistance include, but are not limited to, Y1H, Y1I, S3Q, S3H,S3N, G4Q, G4H, G4N, K5N, K5Q, L61, L6V, E7Q, E7H, E7N, E8Q, E8H, E8N,F91, F9V, V10Q, V10H, V10N, G12Q, G12H, G12N, L141, L14V, E15Q, E15H,E15N, R16H, R16Q, E17Q, E17H, E17N, M191, M19V, E20Q, E20H, E20N, E21Q,E21H, E21N, K22N, K22Q, S24Q, S24H, 524N, F251, F25V, E26Q, E26H, E26N,E27Q, E27H, E27N, A28Q, A28H, A28N, R29H, R29Q, E30Q, E30H, E30N, V31Q,V31H, V31N, F321, F32V, E33Q, E33H, E33N, T35Q, T35H, T35N, E36Q, E36H,E36N, R37H, R37Q, T38Q, T38H, T38N, T39Q, T39H, T39N, E40Q, E40H, E40N,F411, F41V, W42S, W42H, K43N, K43Q, Y45H, Y451, V46Q, V46H, V46N, D47N,D47Q, G48Q, G48H, G48N, D49N, D49Q, E52Q, E52H, E52N, S53Q, S53H, S53N,P55A, P55S, L571, L57V, N58Q, N58S, G59Q, G59H, G59N, G60Q, G60H, G60N,S61Q, S61H, S61N, K63N, K63Q, D64N, D64Q, D65N, D65Q, I66Q, I66H, I66N,S68Q, S68H, S68N, Y69H, Y691, E70Q, E70H, E70N, W72S, W72H, P74A, P74S,F751, F75V, G76Q, G76H, G76N, F771, F77V, E78Q, E78H, E78N, G79Q, G79H,G79N, K80N, K80Q, E83Q, E83H, E83N, L841, L84V, D85N, D85Q, V86Q, V86H,V86N, T87Q, T87H, T87N, I90Q, 190H, 190N, K91N, K91Q, N92Q, N92S, G93Q,G93H, G93N, R94H, R94Q, E96Q, E96H, E96N, F981, F98V, K100N, K100Q,S102Q, S102H, S102N, A103Q, A103H, A103N, D104N, D104Q, K106N, K106Q,V107Q, V107H, V107N, V108Q, V108H, V108N, S110Q, S110H, S110N, T112Q,T112H, T112N, E113Q, E113H, E113N, G114Q, G114H, G114N, Y115H, Y115I,R116H, R116Q, L117I, L117V, A118Q, A118H, A118N, E119Q, E119H, E119N,K122N, K122Q, S123Q, S123H, S123N, E125Q, E125H, E125N, P126A, P126S,A127Q, A127H, A127N, V128Q, V128H, V128N, P129A, P129S, P131A, P131S,G133Q, G133H, G133N, R134H, R134Q, V135Q, V135H, V135N, S136Q, S136H,S136N, V137Q, V137H, V137N, S138Q, S138H, S138N, T140Q, T140H, T140N,S141Q, S141H, S141N, K142N, K142Q, L143I, L143V, T144Q, T144H, T144N,R145H, R145Q, A146Q, A146H, A146N, E147Q, E147H, E147N, T148Q, T148H,T148N, V149Q, V149H, V149N, P151A, P151S, D152N, D152Q, V153Q, V153H,V153N, D154N, D154Q, Y155H, Y155I, V156Q, V156H, V156N, S158Q, S158H,S158N, T159Q, T159H, T159N, E160Q, E160H, E160N, A161Q, A161H, A161N,E162Q, E162H, E162N, T163Q, T163H, T163N, I164Q, 1164H, 1164N, L165I,L165V, L165Q, L165H, D166N, D166Q, I168Q, I168H, I168N, T169Q, T169H,T169N, S171Q, S171H, S171N, T172Q, T172H, T172N, S174Q, S174H, S174N,F175I, F175V, F175H, D177N, D177Q, F178I, F178V, F178H, T179Q, T179H,T179N, R180H, R180Q, V181Q, V181H, V181N, V182Q, V182H, V182N, G183Q,G183H, G183N, G184Q, G184H, G184N, E185Q, E185H, E185N, D186N, D186Q,A187Q, A187H, A187N, K188N, K188Q, P189A, P189S, G190Q, G190H, G190N,F192I, F192V, F1921H, P193A, P193S, W194S, W194H, W194I, V196Q, V196H,V196N, V197Q, V197H, V197N, L198I, L198V, L198Q, L198H, N199Q, N199S,G200Q, G200H, G200N, K201N, K201Q, V202Q, V202H, V202N, D203N, D203Q,A204Q, A204H, A204N, F205I, F205V, G207Q, G207H, G207N, G208Q, G208H,G208N, S209Q, S209H, S209N, I210Q, 1210H, 1210N, V211Q, V211H, V211N,E213Q, E213H, E213N, K214N, K214Q, W215S, W215H, I216Q, I216H, I216N,V217Q, V217H, V217N, T218Q, T218H, T218N, A219Q, A219H, A219N, A220Q,A220H, A220N, V223Q, V223H, V223N, E224Q, E224H, E224N, T225Q, T225H,T225N, G226Q, G226H, G226N, V227Q, V227H, V227N, K228N, K228Q, I229Q,I229H, I229N, T230Q, T230H, T230N, V231Q, V231H, V231N, V232Q, V232H,V232N, A233Q, A233H, A233N, G234Q, G234H, G234N, E235Q, E235H, E235N,I238Q, I238H, I238N, E239Q, E239H, E239N, E240Q, E240H, E240N, T241Q,T241H, T241N, E242Q, E242H, E242N, T244Q, T244H, T244N, E245Q, E245H,E245N, K247N, K247Q, R248H, R248Q, V250Q, V250H, V250N, I251Q, 1251H,I251N, R252H, R252Q, 1253Q, 1253H, 1253N, 1254Q, 1254H, 1254N, P255A,P255S, Y259H, Y259I, A261Q, A261H, A261N, A262Q, A262H, A262N, I263Q,I263H, I263N, K265N, K265Q, Y266H, Y266I, D269N, D269Q, I270Q, 1270H,1270N, A271Q, A271H, A271N, L272I, L272V, L273I, L273V, E274Q, E274H,E274N, L275I, L275V, D276N, D276Q, E277Q, E277H, E277N, P278A, P278S,L279I, L279V, V280Q, V280H, V280N, L281I, L281V, S283Q, S283H, S283N,Y284H, Y284I, V285Q, V285H, V285N, T286Q, T286H, T286N, P287A, P287S,I288Q, I288H, 1288N, 1290Q, 1290H, 1290N, A291Q, A291H, A291N, D292N,D292Q, K293N, K293Q, E294Q, E294H, E294N, Y295H, Y295I, T296Q, T296H,T296N, I298Q, 1298H, 1298N, F299I, F299V, L300I, L300V, K301N, K301Q,F302I, F302V, G303Q, G303H, G303N, S304Q, S304H, S304N, G305Q, G305H,G305N, Y306H, Y306I, V307Q, V307H, V307N, S308Q, S308H, S308N, G309Q,G309H, G309N, W310S, W310H, G311Q, G311H, G311N, R312H, R312Q, V313Q,V313H, V313N, F314I, F314V, K316N, K316Q, G317Q, G317H, G317N, R318H,R318Q, S319Q, S319H, S319N, A320Q, A320H, A320N, L321I, L321V, V322Q,V322H, V322N, L323I, L323V, Y325H, Y325I, L326I, L326V, R327H, R327Q,V328Q, V328H, V328N, P329A, P329S, L330I, L330V, V331Q, V331H, V331N,D332N, D332Q, R333H, R333Q, A334Q, A334H, A334N, T335Q, T335H, T335N,L337I, L337V, R338H, R338Q, S339Q, S339H, S339N, T340Q, T340H, T340N,K341N, K341Q, F342I, F342V, T343Q, T343H, T343N, I344Q, I344H, 1344N,Y345H, Y345I, M348I, M348V, F349I, F349V, A351Q, A351H, A351N, G352Q,G352H, G352N, F353I, F353V, E355Q, E355H, E355N, G356Q, G356H, G356N,G357Q, G357H, G357N, R358H, R358Q, D359N, D359Q, S360Q, S360H, S360N,G363Q, G363H, G363N, D364N, D364Q, S365Q, S365H, S365N, G366Q, G366H,G366N, G367Q, G367H, G367N, P368A, P368S, V370Q, V370H, V370N, T371Q,T371H, T371N, E372Q, E372H, E372N, V373Q, V373H, V373N, E374Q, E374H,E374N, G375Q, G375H, G375N, T376Q, T376H, T376N, S377Q, S377H, S377N,F378I, F378V, L379I, L379V, T380Q, T380H, T380N, G381Q, G381H, G381N,I382Q, I382H, I382N, 1383Q, 1383H, 1383N, S384Q, S384H, S384N, W385S,W385H, G386Q, G386H, G386N, E387Q, E387H, E387N, E388Q, E388H, E388N,A390Q, A390H, A390N, M391I, M391V, K392N, K392Q, G393Q, G393H, G393N,K394N, K394Q, Y395H, Y395I, G396Q, G396H, G396N, I397Q, 1397H, 1397N,Y398H, Y398I, T399Q, T399H, T399N, K400N, K400Q, V401Q, V401H, V401N,S402Q, S402H, S402N, R403H, R403Q, Y404H, Y404I, V405Q, V405H, V405N,W407S, W407H, I408Q, 1408H, 1408N, K409N, K409Q, E410Q, E410H, E410N,K411N, K411Q, T412Q, T412H, T412N, K413N, K413Q, L414I, L414V, T415Q,T415H, and T415N (numbering corresponding to a mature FIX polypeptideset forth in SEQ ID NO:3).

f. Modifications to Reduce Immunogenicity

Further modifications to a modified FIX polypeptide provided herein caninclude modifications of at least one amino acid residue resulting in asubstantial reduction in activity of or elimination of one or more Tcell epitopes from the protein, i.e. deimmunization of the polypeptide.One or more amino acid modifications at particular positions within anyof the MHC class II ligands can result in a deimmunized FIX polypeptidewith reduced immunogenicity when administered as a therapeutic to asubject, such as for example, a human subject. For example, any one ormore modifications disclosed in U.S. Patent Publication No. 20040254106can be included in the modified FIX polypeptide provided herein toreduce immunogenicity.

Exemplary amino acid modifications that can contribute to reducedimmunogenicity of a FIX polypeptide include any one or more amino acidmodifications corresponding to any one or more of the followingmodifications: Y1A, Y1C, Y1D, Y1E, Y1G, Y1H, Y1K, Y1N, Y1P, Y1Q, Y1R,Y1S, Y1T, S3T, L6A, L6C, L6D, L6E, L6G, L6H, L6K, L6N, L6P, L6Q, L6R,L6S, L6T, L6M, F9A, F9C, F9D, F9E, F9G, F9H, F9K, F9N, F9P, F9Q, F9R,F9S, F9T, F91, F9M, F9W, V10A, V10C, V10D, V10E, V10G, V10H, V10K, V10N,V10P, V10Q, V10R, V10S, V10T, V10F, V10I, V10M, V10W, V10Y, Q11A, Q11C,Q11G, Q11P, G12D, G12E, G12G, G12H, G12K, G12N, G12P, G12Q, G12R, G12S,G12T, N13A, N13C, N13G, N13H, N13P, N13T, L14A, L14C, L14D, L14E, L14G,L14H, L14K, L14N, L14P, L14Q, L14R, L14S, L14T, L14F, L141, L14M, L14V,L14W, L14Y, E15D, E15H, E15P, R16A, R16C, R16G, R16P, R16T, E17A, E17C,E17G, E17P, E17T, C18D, C18E, C18G, C18H, C18K, C18N, C18P, C18Q, C18R,C18S, C18T, M19A, M19C, M19D, M19E, M19G, M19H, M19K, M19N, M19P, M19Q,M19R, M19S, M19T, M19F, M191, M19M, M19V, M19W, M19Y, E20A, E20C, E20G,E20P, E20T, E21A, E21C, E21G, E21P, K22H, K22P, K22T, S24H, S24P, F25A,F25C, F25D, F25E, F25G, F25H, F25K, F25N, F25P, F25Q, F25R, F25S, F25T,F251, F25M, F25W, F25Y, E26A, E26C, E26G, E26P, E27A, E27C, E27G, E27H,E27P, E27S, E27T, A28C, A28D, A28E, A28G, A28H, A28K, A28N, A28P, A28Q,A28R, A28S, A28T, R29A, R29C, R29G, R29P, E30D, E30H, E30P, V31A, V31C,V31D, V31E, V31G, V31H, V31K, V31N, V31P, V31Q, V31R, V31S, V31T, V31F,V311, V31W, V31Y, F32A, F32C, F32D, F32E, F32G, F32H, F32K, F32N, F32P,F32Q, F32R, F32S, F32T, E33H, E33N, E33P, E33Q, E33S, E33T, T35A, T35C,T35G, T35P, F41A, F41C, F41D, F41E, F41G, F41H, F41K, F41N, F41P, F41Q,F41R, F41S, F41T, F41M, F41W, F41Y, W42A, W42C, W42D, W42E, W42G, W42H,W42K, W42N, W42P, W42Q, W42R, W425, W42T, K43A, K43C, K43G, K43P, Q44P,Q44T, Q44, Y45A, Y45C, Y45D, Y45E, Y45G, Y45H, Y45K, Y45N, Y45P, Y45Q,Y45R, Y45S, Y45T, V46A, V46C, V46D, V46E, V46G, V46H, V46K, V46N, V46P,V46Q, V46R, V46S, V46T, V46F, V461, V46M, V46W, V46Y, D47A, D47C, D47G,D47H, D47P, D47T, G48D, G48E, G48P, G48T, D49H, D49P, D49Q, D49T, Q50A,Q50C, Q50D, Q50G, Q50H, Q50P, Q50T, C51D, C51E, C51G, C51H, C51K, C51N,C51P, C51Q, C51R, C51S, C51T, E52P, E52T, S53A, S53C, S53G, S53H, S53P,S53T, N54H, N54P, N54T, L57A, L57C, L57D, L57E, L57G, L57H, L57K, L57N,L57P, L57Q, L57R, L57S, L57T, L57F, L571, L57M, L57W, L57Y, G60C, G60D,G60H, G60P, G60T, C62D, C62H, C62P, K63T, D65H, D65T, I66A, I66C, I66D,166E, 166G, I66H, I66K, I66N, I66P, I66Q, 166R, 166S, 166T, 166M, 166W,166Y, Y69A, Y69C, Y69D, Y69E, Y69G, Y69H, Y69K, Y69N, Y69P, Y69Q, Y69R,Y69S, Y69T, C71H, C71P, W72A, W72C, W72D, W72E, W72G, W72H, W72K, W72N,W72P, W72Q, W72R, W72S, W72T, W721, W72Y, F75A, F75C, F75D, F75E, F75G,F75H, F75K, F75N, F75P, F75Q, F75R, F75S, F75T, F77A, F77C, F77D, F77E,F77G, F77H, F77K, F77N, F77P, F77Q, F77R, F77S, F77T, L84A, L84C, L84D,L84E, L84G, L84H, L84K, L84N, L84P, L84Q, L84R, L84S, L84T, L84M, L84W,L84Y, V86A, V86C, V86D, V86E, V86G, V86H, V86K, V86N, V86P, V86Q, V86R,V86S, V86T, 190A, 190C, 190D, 190E, 190G, 190H, 190K, 190N, I90P, I90Q,190R, 190S, 190T, 190M, 190W, K91A, K91C, K91G, K91P, N92A, N92C, N92G,N92P, N92T, G93D, G93E, G93H, G93K, G93N, G93P, G93Q, G93R, G93S, G93T,R94A, R94C, R94G, R94P, C95D, C95E, C95G, C95H, C95K, C95N, C95P, C95Q,C95R, C95S, C95T, E96P, E96T, Q97A, Q97C, Q97G, Q97P, F98A, F98C, F98D,F98E, F98G, F98H, F98K, F98N, F98P, F98Q, F98R, F98S, F98T, F98M, F98W,F98Y, K100A, K100C, K100G, K100P, N101H, N101T, A103D, A103E, A103H,A103K, A103N, A103P, A103Q, A103R, A103S, A103T, D104T, K106H, K106P,K106T, V107A, V107C, V107D, V107E, V107G, V107H, V107K, V107N, V107P,V107Q, V107R, V107S, V107T, V108A, V108C, V108D, V108E, V108G, V108H,V108K, V108N, V108P, V108Q, V108R, V108S, V108T, V108F, V108M, V108W,V108Y, S110A, S110C, S110G, S110P, C111D, C111E, C111H, C111K, C111N,C111P, C111Q, C111R, C111S, C111T, T112A, T112C, T112G, T112P, E113D,E113H, E113P, G114D, G114E, G114H, G114K, G114N, G114P, G114Q, G114R,G114S, G114T, Y115A, Y115C, Y115D, Y115E, Y115G, Y115H, Y115K, Y115N,Y115P, Y115Q, Y115R, Y115S, Y115T, Y115M, Y115W, R116P, R116T, L117A,L117C, L117D, L117E, L117G, L117H, L117K, L117N, L117P, L117Q, L117R,L117S, L117T, A118D, A118E, A118H, A118K, A118N, A118P, A118Q, A118R,A118S, A118T, N120D, N120H, N120P, Q121T, S123H, S123T, V128A, V128C,V128D, V128E, V128G, V128H, V128K, V128N, V128P, V128Q, V128R, V128S,V128T, F130A, F130C, F130D, F130E, F130G, F130H, F130K, F130N, F130P,F130Q, F130R, F130S, F130T, V135A, V135C, V135D, V135E, V135G, V135H,V135K, V135N, V135P, V135Q, V135R, V135S, V135T, V135W, V135Y, V137A,V137C, V137D, V137E, V137G, V137H, V137K, V137N, V137P, V137Q, V137R,V137S, V137T, V137M, V137W, V137Y, S138H, S138T, T140D, T140H, S141T,K142H, K142P, L143A, L143C, L143D, L143E, L143G, L143H, L143K, L143N,L143P, L143Q, L143R, L143S, L143T, L143F, L1431, L143M, L143V, L143W,L143Y, R145H, R145P, R145T, A146P, A146T, T148H, T148P, V149A, V149C,V149D, V149E, V149G, V149H, V149K, V149N, V149P, V149Q, V149R, V149S,V149T, V149F, V149I, V149M, V149W, V149Y, F150A, F150C, F150D, F150E,F150G, F150H, F150K, F150N, F150P, F150Q, F150R, F150S, F150T, F150M,F150W, F150Y, D152A, D152C, D152G, D152P, D152S, D152T, V153A, V153C,V153D, V153E, V153G, V153H, V153K, V153N, V153P, V153Q, V153R, V153S,V153T, V153F, V1531, V153M, V153W, V153Y, D154A, D154C, D154G, D154P,D154Q, D154S, Y155A, Y155C, Y155D, Y155E, Y155G, Y155H, Y155K, Y155N,Y155P, Y155Q, Y155R, Y155S, Y155T, Y155M, Y155V, Y155W, V156A, V156C,V156D, V156E, V156G, V156H, V156K, V156N, V156P, V156Q, V156R, V156S,V156T, V1561, V156M, V156W, V156Y, N157A, N157C, N157G, N157H, N157P,N157Q, N157T, S158H, S158P, S158T, T159A, T159C, T159G, T159P, E160A,E160C, E160G, E160P, A161C, A161D, A161E, A161H, A161K, A161N, A161P,A161Q, A161R, A161S, A161T, E162P, E162T, T163A, T163C, T163G, T163P,I164A, I164C, I164D, 1164E, I164G, I164H, I164K, I164N, I164P, 1164Q,I164R, I164S, I164T, L165A, L165C, L165D, L165E, L165G, L165H, L165K,L165N, L165P, L165Q, L165R, L165S, L165T, L165M, L165W, L165Y, 1168A,I168C, I168D, 1168E, I168G, I168H, I168K, I168N, I168P, I168Q, I168R,I168S, I168T, F175A, F175C, F175D, F175E, F175G, F175H, F175K, F175N,F175P, F175Q, F175R, F175S, F175T, F178A, F178C, F178D, F178E, F178G,F178H, F178K, F178N, F178P, F178Q, F178R, F178S, F178T, F178M, F178W,F178Y, T179A, T179C, T179G, T179P, R180A, R180C, R180D, R180G, R180H,R180P, V181A, V181C, V181D, V181E, V181G, V181H, V181K, V181N, V181P,V181Q, V181R, V181S, V181T, V181F, V1811, V181M, V181W, V181Y, V182A,V182C, V182D, V182E, V182G, V182H, V182K, V182N, V182P, V182Q, V182R,V182S, V182T, V182F, V182I, V182M, V182W, V182Y, G183D, G183E, G183H,G183K, G183N, G183P, G183Q, G183S, G183T, G184D, G184E, G184H, G184K,G184N, G184P, G184Q, G184R, G184S, G184T, E185A, E185C, E185G, E185H,E185P, E185T, D186A, D186C, D186G, D186H, D186P, D186T, A187C, A187D,A187E, A187G, A187H, A187K, A187N, A187P, A187Q, A187R, A187S, A187T,K188A, K188C, K188G, K188H, K188P, K188T, G190D, G190E, G190H, G190K,G190N, G190P, G190Q, G190R, G190S, G190T, F192A, F192C, F192D, F192E,F192G, F192H, F192K, F192N, F192P, F192Q, F192R, F192S, F192T, F192W,F192Y, W194A, W194C, W194D, W194E, W194G, W194H, W194K, W194N, W194P,W194Q, W194R, W194S, W194T, Q195H, Q195P, Q195T, V196A, V196C, V196D,V196E, V196G, V196H, V196K, V196N, V196P, V196Q, V196R, V196S, V196T,V196F, V196I, V196M, V196W, V196Y, V197A, V197C, V197D, V197E, V197G,V197H, V197K, V197N, V197P, V197Q, V197R, V197S, V197T, V197F, V197I,V197M, V197W, V197Y, L198A, L198C, L198D, L198E, L198G, L198H, L198K,L198N, L198P, L198Q, L198R, L198S, L198T, L1981, L198Y, N199A, N199C,N199G, N199H, N199P, N199S, N199T, G200P, G200T, K201A, K201C, K201D,K201E, K201G, K201H, K201N, K201P, K201Q, K201S, K201T, V202A, V202C,V202D, V202E, V202G, V202H, V202K, V202N, V202P, V202Q, V202R, V202S,V202T, V202F, V2021, V202M, V202W, V202Y, D203A, D203C, D203G, D203P,D203T, A204C, A204D, A204E, A204G, A204H, A204K, A204N, A204P, A204Q,A204R, A204S, A204T, F205A, F205C, F205D, F205E, F205G, F205H, F205K,F205N, F205P, F205Q, F205R, F205S, F205T, F205M, F205V, F205W, F205Y,G207H, G207P, G208C, G208D, G208E, G208H, G208K, G208N, G208P, G208Q,G208R, G208S, G208T, S209A, S209C, S209G, S209P, I210A, I210C, I210D,I210E, I210G, I210H, I210K, I210N, I210P, I210Q, I210R, I210S, I210T,I210F, I210W, I210Y, V211A, V211C, V211D, V211E, V211G, V211H, V211K,V211N, V211P, V211Q, V211R, V211S, V211T, V211F, V211I, V211M, V211W,N212A, N212C, N212G, N212P, E213H, E213P, E213S, E213T, K214T, W215A,W215C, W215D, W215E, W215G, W215H, W215K, W215N, W215P, W215Q, W215R,W215S, W215T, I216A, I216C, I216D, I216E, I216G, I216H, I216K, I216N,I216P, I216Q, I216R, I216S, I216T, V217A, V217C, V217D, V217E, V217G,V217H, V217K, V217N, V217P, V217Q, V217R, V217S, V217T, V217I, V217Y,A219H, A219P, A219T, V223A, V223C, V223D, V223E, V223G, V223H, V223K,V223N, V223P, V223Q, V223R, V223S, V223T, V223M, V223W, V223Y, G226P,V227A, V227C, V227D, V227E, V227G, V227H, V227K, V227N, V227P, V227Q,V227R, V227S, V227T, V227F, V227I, V227M, V227W, V227Y, K228A, K228C,K228G, K228H, K228P, I229A, I229C, I229D, I229E, I229G, I229H, I229K,I229N, I229P, I229Q, I229R, I229S, I229T, I229M, I229W, I229Y, T230A,T230C, T230G, T230P, V231A, V231C, V231D, V231E, V231G, V231H, V231K,V231N, V231P, V231Q, V231R, V231S, V231T, V232A, V232C, V232D, V232E,V232G, V232H, V232K, V232N, V232P, V232Q, V232R, V232S, V232T, V232F,V2321, V232M, V232W, V232Y, A233C, A233D, A233E, A233G, A233H, A233K,A233N, A233P, A233Q, A233R, A233S, A233T, A233V, G234D, G234E, G234H,G234K, G234N, G234P, G234Q, G234R, G234S, G234T, E235H, E235N, E235P,E235Q, E235S, E235T, H236A, H236C, H236G, H236P, N237A, N237C, N237G,N237P, N237T, I238A, I238C, I238D, I238E, I238G, I238H, I238K, I238N,I238P, I238Q, I238R, I238S, I238T, E239A, E239C, E239G, E239P, E240H,E240T, V250A, V250C, V250D, V250E, V250G, V250H, V250K, V250N, V250P,V250Q, V250R, V250S, V250T, V250M, V250W, V250Y, I251A, I251C, I251D,I251E, I251G, I251H, I251K, I251N, I251P, I251Q, I251R, I251S, I251T,I253A, I253C, I253D, I253E, I253G, I253H, I253K, I253N, I253P, I253Q,I253R, I253S, I253T, I253M, I253W, I253Y, I254A, I254C, I254D, I254E,I254G, I254H, I254K, I254N, I254P, I254Q, I254R, I254S, I254T, P255H,H256P, H256T, H257A, H257C, H257G, H257P, N258P, N258T, Y259A, Y259C,Y259D, Y259E, Y259G, Y259H, Y259K, Y259N, Y259P, Y259Q, Y259R, Y259S,Y259T, Y259M, Y259W, N260A, N260C, N260G, N260P, A261D, A261E, A261H,A261K, A261N, A261P, A261Q, A261R, A261S, A261T, A262C, A262D, A262E,A262G, A262H, A262K, A262N, A262P, A262Q, A262R, A262S, A262T, I263A,I263C, I263D, I263E, I263G, I263H, I263K, I263N, I263P, I263Q, I263R,I263S, I263T, I263M, I263V, I263W, I263Y, N264A, N264C, N264D, N264G,N264H, N264P, K265A, K265C, K265G, K265H, K265P, Y266A, Y266C, Y266D,Y266E, Y266G, Y266H, Y266K, Y266N, Y266P, Y266Q, Y266R, Y266S, Y266T,Y266M, Y266W, N267A, N267C, N267G, N267H, N267P, N267T, H268P, D269A,D269C, D269E, D269G, D269H, D269N, D269P, D269Q, D269S, D269T, I270A,I270C, I270D, I270E, I270G, I270H, I270K, I270N, I270P, I270Q, I270R,I270S, I270T, I270M, I270W, A271C, A271D, A271E, A271G, A271H, A271K,A271N, A271P, A271Q, A271R, A271S, A271T, L272A, L272C, L272D, L272E,L272G, L272H, L272K, L272N, L272P, L272Q, L272R, L272S, L272T, L272F,L273A, L273C, L273D, L273E, L273G, L273H, L273K, L273N, L273P, L273Q,L273R, L273S, L273T, L273F, L273I, L273M, L273V, L273W, L273Y, E274A,E274C, E274G, E274P, E274T, L275A, L275C, L275D, L275E, L275G, L275H,L275K, L275N, L275P, L275Q, L275R, L275S, L275T, L275W, L275Y, D276P,D276S, D276T, E277A, E277C, E277G, E277P, P278T, L279A, L279C, L279D,L279E, L279G, L279H, L279K, L279N, L279P, L279Q, L279R, L279S, L279T,L279I, L279Y, V280A, V280C, V280D, V280E, V280G, V280H, V280K, V280N,V280P, V280Q, V280R, V280S, V280T, V280F, V2801, V280W, V280Y, L281A,L281C, L281D, L281E, L281G, L281H, L281K, L281N, L281P, L281Q, L281R,L281S, L281T, L281F, L281I, L281V, L281W, L281Y, S283A, S283C, S283G,S283P, Y284A, Y284C, Y284D, Y284E, Y284G, Y284H, Y284K, Y284N, Y284P,Y284Q, Y284R, Y284S, Y284T, Y284M, V285A, V285C, V285D, V285E, V285G,V285H, V285K, V285N, V285P, V285Q, V285R, V285S, V285T, V285M, V285W,V285Y, T286A, T286C, T286G, T286P, I288A, I288C, I288D, I288E, I288G,I288H, I288K, I288N, I288P, I288Q, I288R, I288S, I288T, C289D, C289H,C289P, I290A, I290C, I290D, I290E, I290G, I290H, I290K, I290N, I290P,I290Q, I290R, I290S, I290T, I290Y, A291D, A291E, A291H, A291K, A291N,A291P, A291Q, A291R, A291S, A291T, D292A, D292C, D292G, D292P, D292T,K293H, K293P, K293T, Y295A, Y295C, Y295D, Y295E, Y295G, Y295H, Y295K,Y295N, Y295P, Y295Q, Y295R, Y295S, Y295T, Y295W, T296A, T296C, T296G,T296P, N297A, N297C, N297G, N297P, I298A, I298C, I298D, I298E, I298G,I298H, I298K, I298N, I298P, I298Q, I298R, I298S, I298T, F299A, F299C,F299D, F299E, F299G, F299H, F299K, F299N, F299P, F299Q, F299R, F299S,F299T, L300A, L300C, L300D, L300E, L300G, L300H, L300K, L300N, L300P,L300Q, L300R, L300S, L300T, L300F, L300I, L300M, L300V, L300W, L300Y,K301A, K301C, K301G, K301P, K301T, F302A, F302C, F302D, F302E, F302G,F302H, F302K, F302N, F302P, F302Q, F302R, F302S, F302T, G303H, G303P,G303T, S304A, S304C, S304G, S304P, S304T, G305D, G305E, G305H, G305N,G305P, G305Q, G305S, G305T, Y306A, Y306C, Y306D, Y306E, Y306G, Y306H,Y306K, Y306N, Y306P, Y306Q, Y306R, Y306S, Y306T, V307A, V307C, V307D,V307E, V307G, V307H, V307K, V307N, V307P, V307Q, V307R, V307S, V307T,S308P, S308T, W310A, W310C, W310D, W310E, W310G, W310H, W310K, W310N,W310P, W310Q, W310R, W310S, W310T, G311H, V313A, V313C, V313D, V313E,V313G, V313H, V313K, V313N, V313P, V313Q, V313R, V313S, V313T, F314A,F314C, F314D, F314E, F314G, F314H, F314K, F314N, F314P, F314Q, F314R,F314S, F314T, F314M, F314W, F314Y, H315A, H315C, H315G, H315P, K316A,K316C, K316G, K316P, G317C, G317D, G317E, G317H, G317K, G317N, G317P,G317Q, G317R, G317S, G317T, R318A, R318C, R318G, R318P, S319D, S319H,S319N, S319P, S319Q, A320C, A320D, A320E, A320G, A320H, A320K, A320N,A320P, A320Q, A320R, A320S, A320T, L321A, L321C, L321D, L321E, L321G,L321H, L321K, L321N, L321P, L321Q, L321R, L321S, L321T, V322A, V322C,V322D, V322E, V322G, V322H, V322K, V322N, V322P, V322Q, V322R, V322S,V322T, V322W, V322Y, L323A, L323C, L323D, L323E, L323G, L323H, L323K,L323N, L323P, L323Q, L323R, L323S, L323T, L323F, L323I, L323M, L323V,L323W, L323Y, Q324A, Q324C, Q324G, Q324P, Y325A, Y325C, Y325D, Y325E,Y325G, Y325H, Y325K, Y325N, Y325P, Y325Q, Y325R, Y325S, Y325T, Y325W,L326A, L326C, L326D, L326E, L326G, L326H, L326K, L326N, L326P, L326Q,L326R, L326S, L326T, L326F, L326I, L326M, L326V, L326W, L326Y, R327A,R327C, R327G, R327H, R327P, V328A, V328C, V328D, V328E, V328G, V328H,V328K, V328N, V328P, V328Q, V328R, V328S, V328T, V328F, V3281, V328M,V328W, V328Y, L330A, L330C, L330D, L330E, L330G, L330H, L330K, L330N,L330P, L330Q, L330R, L330S, L330T, L330F, L330I, L330V, L330W, L330Y,V331A, V331C, V331D, V331E, V331G, V331H, V331K, V331N, V331P, V331Q,V331R, V331S, V331T, V331F, V331I, V331M, V331W, V331Y, D332A, D332C,D332G, D332P, R333A, R333C, R333D, R333E, R333G, R333H, R333N, R333P,R333Q, R333R, R333S, R333T, A334C, A334D, A334E, A334G, A334H, A334K,A334N, A334P, A334Q, A334R, A334S, A334T, T335A, T335C, T335G, T335P,C336D, C336E, C336H, C336K, C336N, C336P, C336Q, C336R, C336S, C336T,L337A, L337C, L337D, L337E, L337G, L337H, L337K, L337N, L337P, L337Q,L337R, L337S, L337T, R338A, R338C, R338G, R338P, S339P, S339T, K341A,K341C, K341G, K341P, F342A, F342C, F342D, F342E, F342G, F342H, F342K,F342N, F342P, F342Q, F342R, F342S, F342T, F342M, F342W, T343A, T343C,T343G, T343P, I344A, I344C, I344D, I344E, I344G, I344H, I344K, I344N,I344P, I344Q, I344R, I344S, I344T, Y345A, Y345C, Y345D, Y345E, Y345G,Y345H, Y345K, Y345N, Y345P, Y345Q, Y345R, Y345S, Y345T, Y345M, Y345W,N346A, N346C, N346G, N346P, N347H, N347P, M348A, M348C, M348D, M348E,M348G, M348H, M348K, M348N, M348P, M348Q, M348R, M348S, M348T, F349A,F349C, F349D, F349E, F349G, F349H, F349K, F349N, F349P, F349Q, F349R,F349S, F349T, F349I, F349M, F349W, F349Y, C350D, C350H, C350P, C350T,A351E, A351H, A351N, A351P, A351Q, A351R, A351S, A351T, G352A, G352C,G352P, F353A, F353C, F353D, F353E, F353G, F353H, F353K, F353N, F353P,F353Q, F353R, F353S, F353T, F353I, F353M, F353W, H354A, H354C, H354G,H354P, E355A, E355C, E355D, E355G, E355H, E355K, E355N, E355P, E355Q,E355S, E355T, G356D, G356E, G356H, G356K, G356N, G356P, G356Q, G356R,G356S, G356T, G357D, G357E, G357H, G357K, G357N, G357P, G357Q, G357R,G357S, G357T, R358D, R358E, R358H, R358K, R358N, R358P, R358Q, R358R,R358S, R358T, D359A, D359C, D359G, D359P, D359Q, D359S, D359T, S360A,S360C, S360G, S360P, C361D, C361E, C361H, C361K, C361N, C361P, C361Q,C361R, C361S, C361T, V370A, V370C, V370D, V370E, V370G, V370H, V370K,V370N, V370P, V370Q, V370R, V370S, V370T, V370W, V370Y, V373A, V373C,V373D, V373E, V373G, V373H, V373K, V373N, V373P, V373Q, V373R, V373S,V373T, V373F, V3731, V373M, V373W, E374A, E374C, E374G, E374P, G375H,S377A, S377C, S377G, S377P, F378A, F378C, F378D, F378E, F378G, F378H,F378K, F378N, F378P, F378Q, F378R, F378S, F378T, F378W, L379A, L379C,L379D, L379E, L379G, L379H, L379K, L379N, L379P, L379Q, L379R, L379S,L379T, L379I, L379M, L379W, L379Y, T380A, T380C, T380G, T380P, G381D,G381E, G381H, G381K, G381N, G381P, G381Q, G381R, G381S, G381T, I382A,I382C, I382D, I382E, I382G, I382H, I382K, I382N, I382P, I382Q, I382R,I382S, I382T, I382M, I382W, I382Y, I383A, I383C, I383D, I383E, I383G,I383H, I383K, I383N, I383P, I383Q, I383R, I383S, I383T, S384A, S384C,S384G, S384P, W385A, W385C, W385D, W385E, W385G, W385H, W385K, W385N,W385P, W385Q, W385R, W385S, W385T, W385M, E387A, E387C, E387G, E387H,E387P, E387T, E388H, E388N, E388P, E388Q, E388T, A390C, A390D, A390E,A390G, A390H, A390K, A390N, A390P, A390Q, A390R, A390S, M391A, M391C,M391D, M391E, M391G, M391H, M391K, M391N, M391P, M391Q, M391R, M391S,M391T, M391F, M391I, M391W, M391Y, K392A, K392C, K392G, K392P, G393C,G393D, G393E, G393H, G393K, G393N, G393P, G393Q, G393R, G393S, G393T,Y395A, Y395C, Y395D, Y395E, Y395G, Y395H, Y395K, Y395N, Y395P, Y395Q,Y395R, Y395S, Y395T, Y398A, Y398C, Y398D, Y398E, Y398G, Y398H, Y398K,Y398N, Y398P, Y398Q, Y398R, Y398S, Y398T, K400H, V401A, V401C, V401D,V401E, V401G, V401H, V401K, V401N, V401P, V401Q, V401R, V401S, V401T,V401F, V4011, V401M, V401W, V401Y, S402A, S402C, S402G, S402P, R403A,R403C, R403G, R403P, R403T, Y404A, Y404C, Y404D, Y404E, Y404G, Y404H,Y404K, Y404N, Y404P, Y404Q, Y404R, Y404S, Y404T, V405A, V405C, V405D,V405E, V405G, V405H, V405K, V405N, V405P, V405Q, V405R, V405S, V405T,V405W, V405Y, N406F, N406H, N406I, N406L, N406P, N406W, N406Y, W407D,W407E, W407F, W407H, W4071, W407K, W407N, W407P, W407Q, W407R, W407S,W407T, W407Y, I408D, I408E, I408H, I408K, I408N, I408P, I408Q, I408R,I408S, I408T, K409F, K409H, K4091, K409P, K409T, K409V, K409W, K409Y,E410H, K411A, K411C, K411G, K4111, K411P, K411T, K411V, K411W, K411Y orK413T, with numbering corresponding to a mature FIX polypeptide setforth in SEQ ID NO: 3.

g. Exemplary Combination Modifications

Provided herein are modified FIX polypeptides that have two or moremodifications designed to affect one or more properties or activities ofan unmodified FIX polypeptide. In some examples, the two or moremodifications alter two or more properties or activities of the FIXpolypeptide. The modifications can be made to the FIX polypeptides suchthat one or more of glycosylation, resistance to AT-III, resistance toAT-III/heparin, resistance to heparin, catalytic activity, binding toLRP, intrinsic activity, phospholipid binding and/or affinity,resistance to proteases, half-life and interaction with other factors ormolecules, such as FVIIIa and FX, is altered. Typically, the two or moremodifications are combined such that the resulting modified FIXpolypeptide has increased coagulant activity, increased duration ofcoagulant activity, and/or an enhanced therapeutic index compared to anunmodified FIX polypeptide. The modifications can include amino acidsubstitution, insertion or deletion. The increased coagulant activity,increased duration of coagulant activity, and/or an enhanced therapeuticindex of the modified FIX polypeptide containing two or moremodifications can be increased by at least or at least about 1%, 2%, 3%,4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 300%,400%, 500%, or more compared to the activity of the starting orunmodified FIXa polypeptide.

Provided herein are modified FIX polypeptides that contain two or moremodifications that are introduced into an unmodified FIX polypeptide toalter one, two or more activities or properties. The modified FIXpolypeptides can contain 2, 3, 4, 5, 6 or more modifications. Forexample, a modified FIX polypeptide provided herein can contain themodifications to increase glycosylation by incorporating a non-nativeglycosylation site into the primary sequence, such as amino acidsubstitutions D203N and F205T to introduce a non-native glycosylationsite at position 203, and a modification to increase resistance toAT-III/heparin, such as R338E (residues corresponding to a mature FIXpolypeptide set forth in SEQ ID NO:3).

Modified FIX polypeptides provided herein can have two or moremodifications selected solely from those set forth in Tables 3-9. Inother examples, the modified FIX polypeptide contains two or moremodifications where one or more modifications are selected from thoseset forth in Tables 3-9 and one or more modifications are additionalmodifications that are not set forth in Tables 3-9, such as, forexample, modifications described in the art. In some examples, the oneor more additional modifications can be selected from those set forth inSection D.3.a-f, above, such as those that result in increased catalyticactivity, increased resistance to inhibitors, increased affinity and/orbinding to platelets and phospholipids, increased protease resistance,decreased immunogenicity, and those that facilitate conjugation tomoieties, such as PEG moieties.

Non-limiting exemplary combination modifications are provided in Table10. These exemplary combination modifications include two or moremodifications that are designed to alter two or more activities orproperties of a FIX polypeptide, including, but not limited to,increased resistance to AT-III, increased resistance to AT-III/heparin,increased resistance to heparin, increased catalytic activity andaltered glycosylation. Modified FIX polypeptides containing suchcombination modifications can have increased coagulant activity,increased duration of coagulant activity, and/or an enhanced therapeuticindex. In Table 10 below, the sequence identifier (SEQ ID NO) isidentified in which exemplary amino acid sequences of the modified FIXpolypeptide are set forth.

TABLE 10 Mutation Mutation SEQ (Mature FIX Numbering) (ChymotrypsinNumbering) ID NO R318Y/E410N R150Y/E240N 153 R338E/E410N R170E/E240N 154R338E/R403E/E410N R170E/R233E/E240N 155 D203N/F205T/K228N D39N/F41T/K63N157 D203N/F205T/E410N D39N/F41T/E240N 158 D203N/F205T/R338ED39N/F41T/R170E 159 D203N/F205T/R338A D39N/F41T/R170A 160D203N/F205T/R318Y D39N/F41T/R150Y 161 D203N/F205T/R338E/R403ED39N/F41T/R170E/R233E 162 K228N/E410N K63N/E240N 163 K228N/R338EK63N/R170E 164 K228N/R338A K63N/R170A 165 K228N/R318Y K63N/R150Y 166K228N/R338E/R403E K63N/R170E/R233E 167 R403E/E410N R233E/E240N 168R318Y/R338E/E410N R150Y/R170E/E240N 169 K228N/R318Y/E410NK63N/R150Y/E240N 170 R318Y/R403E/E410N R150Y/R233E/E240N 171R318Y/R338E/R403E/E410N R150Y/R170E/R233E/E240N 172D203N/F205T/R318Y/E410N D39N/F41T/R150Y/E240N 173 F314N/K316SF145N/K148S 177 A103N/N105S/K228N A[103]N/N[105]S/K63N 217D104N/K106S/K228N D[104]N/K[106]S/K63N 218 K228N/I251S K63N/I86S 180A103N/N105S/I251S A[103]N/N[105]S/I86S 181 D104N/K106S/I251SD[104]N/K[106]S/I86S 182 A103N/N105S/R318Y/R338E/R403E/A[103]N/N[105]S/R150Y/R170E/ 219 E410N R233E/E240ND104N/K106S/R318Y/R338E/R403E/ D[104]N/K[106]S/R150Y/R170E/ 220 E410NR233E/E240N K228N/R318Y/R338E/R403E/E410N K63N/R150Y/R170E/R233E/E240N221 I251S/R318Y/R338E/R403E/E410N I86S/R150Y/R170E/R233E/E240N 222D104N/K106S/I251S/R318Y/R338E/ D[104]N/K[106]S/I86S/R150Y/ 223R403E/E410N R170E/R233E/E240N D104N/K106S/R318Y/R338E/E410ND[104]N/K[106]S/R150Y/R170E/ 224 E240N I251S/R318Y/R338E/E410NI86S/R150Y/R170E/E240N 225 D104N/K106S/I251S/R318Y/D[104]N/K[106]S/I86S/R150Y/R170E/ 226 R338E/E410N E240NA103N/N105S/K247N/N249S A[103]N/N[105]S/K82N/N84S 178D104N/K106S/K247N/N249S D[104]N/K[106]S/K82N/N84S 179 K228N/K247N/N249SK63N/K82N/N84S 183 A103N/N105S/Y155F A[103]N/N[105]S/Y[155]F 227D104N/K106S/Y155F D[104]N/K[106]S/Y[155]F 228 Y155F/K228N Y[155]F/K63N229 Y155F/I251S Y[155]F/I86S 230 Y155F/K247N/N249S Y[155]F/K82N/N84S 231A103N/N105S/K247N/N249S/R318Y/ A[103]N/N[105]S/K82N/N84S/R150Y/ 232R338E/R403E/E410N R170E/R233E/E240N D104N/K106S/K247N/N249S/D[104]N/K[106]S/K82N/N84S/R150Y/ 233 R318Y/R338E/R403E/E410NR170E/R233E/E240N K228N/K247N/N249S/R318Y/R338E/K63N/K82N/N84S/R150Y/R170E/ 234 R403E/E410N R233E/E240NA103N/N105S/Y155F/R318Y/R338E/ A[103]N/N[105]S/Y[155]F/R150Y/ 235R403E/E410N R170E/R233E/E240N D104N/K106S/Y155F/R318Y/R338E/D[104]N/K[106]S/Y[155]F/R150Y/ 236 R403E/E410N R170E/R233E/E240NY155F/K228N/R318Y/R338E/R403E/ Y[155]F/K63N/R150Y/R170E/R233E/ 237 E410NE240N Y155F/I251S/R318Y/R338E/R403E/ Y[155]F/I86S/R150Y/R170E/R233E/ 238E410N E240N Y155F/K247N/N249S/R318Y/R338E/Y[155]F/K82N/N84S/R150Y/R170E/ 239 R403E/E410N R233E/E240NK247N/N249S/R318Y/R338E/R403E/ K82N/N84S/R150Y/R170E/R233E/ 240 E410NE240N Y155F/R318Y/R338E/R403E/E410N Y[155]F/R150Y/R170E/R233E/E240N 241K247N/N249S/R318Y/R338E/E410N K82N/N84S/R150Y/R170E/E240N 242Y155F/R318Y/R338E/E410N Y[155]F/R150Y/R170E/E240N 243Y155F/K247N/N249S/R318Y/R338E/ Y[155]F/K82N/N84S/R150Y/R170E/ 244 E410NE240N D104N/K106S/Y155F/K228N/K247N/ D[104]N/K[106]S/Y[155]F/K63N/ 245N249S K82N/N84S D104N/K106S/Y155F/K247N/N249SD[104]N/K[106]S/Y[155]F/K82N/ 246 N84S D104N/K106S/Y155F/K228ND[104]N/K[106]S/Y[155]F/K63N 247 Y155F/K228N/K247N/N249SY[155]F/K63N/K82N/N84S 248 D104N/K106S/K228N/K247N/N249SD[104]N/K[106]S/K63N/K82N/N84S 184 R318Y/R338E/R403E/E410SR150Y/R170E/R233E/E240S 249 R318Y/R338E/R403E/E410N/T412VR150Y/R170E/R233E/E240N/T242V 250 R318Y/R338E/R403E/E410N/T412AR150Y/R170E/R233E/E240N/T242A 251 R318Y/R338E/R403E/T412AR150Y/R170E/R233E/T242A 252 R318Y/R338E/E410S R150Y/R170E/E240S 253R318Y/R338E/T412A R150Y/R170E/T242A 254 R318Y/R338E/E410N/T412VR150Y/R170E/E240N/T242V 255 D85N/K228N/R318Y/R338E/R403E/D[85]N/K63N/R150Y/R170E/R233E/ 256 E410N E240NN260S/R318Y/R338E/R403E/E410N N95S/R150Y/R170E/R233E/E240N 257R318Y/R338E/N346D/R403E/E410N R150Y/R170E/N178D/R233E/E240N 258Y155F/N346D Y[155]F/N178D 259 Y155F/R318Y/R338E/N346D/R403E/Y[155]F/R150Y/R170E/N178D/R233E/ 260 E410N E240N Y155F/N260S/N346DY[155]F/N95S/N178D 261 K247N/N249S/N260S K82N/N84S/N95S 262 Y155F/N260SY[155]F/N95S 263 K247N/N249S/N260S/R318Y/R338E/K82N/N84S/N95S/R150Y/R170E/R233E/ 264 R403E/E410N E240ND104N/K106S/N260S/R318Y/R338E/ D[104]N/K[106]S/N95S/R150Y/R170E/ 265R403E/E410N R233E/E240N Y155F/N260S/R318Y/R338E/R403E/Y[155]F/N95S/R150Y/R170E/R233E/ 266 E410N E240NR318Y/R338E/T343R/R403E/E410N R150Y/R170E/T175R/R233E/E240N 267R338E/T343R R170E/T175R 268 D104N/K106S/Y155F/N260SD[104]N/K[106]S/Y[155]F/N95S 269 Y155F/K247N/N249S/N260SY[155]F/K82N/N84S/N95S 270 D104N/K106S/K247N/N249S/N260SD[104]N/K[106]S/K82N/N84S/N95S 271 D104N/K106S/Y155F/K247N/N249S/D[104]N/K[106]S/Y[155]F/K82N/ 272 N260S N84S/N95S D104N/K106S/N260SD[104]N/K[106]S/N95S 185 T343R/Y345T T175R/Y177T 215 R318Y/R338ER150Y/R170E 188 Y259F/K265T/Y345T Y94F/K98T/Y177T 216D104N/K106S/Y155F/K247N/N249S/ D[104]N/K[106]S/Y[155]F/K82N/ 326R318Y/R338E/R403E/E410N N84S/R150Y/R170E/R233E/E240ND104N/K106S/K228N/K247N/N249S/ D[104]N/K[106]S/K63N/K82N/N84S/ 327R318Y/R338E/R403E/E410N R150Y/R170E/R233E/E240NY155F/K228N/K247N/N249S/R318Y/ Y[155]F/K63N/K82N/N84S/R150Y/ 328R338E/R403E/E410N R170E/R233E/E240N Y155F/K247N/N249S/N260S/R318Y/Y[155]F/K82N/N84S/N95S/R150Y/ 329 R338E/R403E/E410N R170E/R233E/E240NY155F/R318Y/R338E/T343R/R403E/ Y[155]F/R150Y/R170E/T175R/R233E/ 330E410N E240N D104N/K106S/R318Y/R338E/T343R/ D[104]N/K[106]S/R150Y/R170E/331 R403E/E410N T175R/R233E/E240N T343R/N346Y T175R/N178Y 332R318Y/R338E/N346Y/R403E/E410N R150Y/R170E/N178Y/R233E/E240N 333R318Y/R338E/T343R/N346Y/R403E/ R150Y/R170E/T175R/N178Y/R233E/ 334 E410NE240N T343R/N346D T175R/N178D 335 R318Y/R338E/T343R/N346D/R403E/R150Y/R170E/T175R/N178D/R233E/ 336 E410N E240NR318Y/R338E/Y345A/R403E/E410N R150Y/R170E/Y177A/R233E/E240N 337R318Y/R338E/Y345A/N346D/R403E/ R150Y/R170E/Y177A/N178D/R233E/ 338 E410NE240N Y155F/K247N/N249S/R318Y/R338E/ Y[155]F/K82N/N84S/R150Y/R170E/ 339R403E R233E K247N/N249S/R318Y/R338E/R403E K82N/N84S/R150Y/R170E/R233E340 Y155F/K247N/N249S/R318Y/R403E/ Y[155]F/K82N/N84S/R150Y/R233E/ 341E410N E240N K247N/N249S/R318Y/R403E/E410N K82N/N84S/R150Y/R233E/E240N342 Y155F/K247N/N249S/R338E/R403E/ Y[155]F/K82N/N84S/R170E/R233E/ 343E410N E240N K247N/N249S/R338E/R403E/E410N K82N/N84S/R170E/R233E/E240N344 R318Y/R338E/T343R/R403E R150Y/R170E/T175R/R233E 345Y155F/R318Y/R338E/T343R/R403E Y[155]F/R150Y/R170E/T175R/R233E 346R318Y/R338E/T343R/E410N R150Y/R170E/T175R/E240N 347Y155F/R318Y/R338E/T343R/E410N Y[155]F/R150Y/R170E/T175R/E240N 348R318Y/T343R/R403E/E410N R150Y/T175R/R233E/E240N 349Y155F/R318Y/T343R/R403E/E410N Y[155]F/R150Y/T175R/R233E/E240N 350R338E/T343R/R403E/E410N R170E/T175R/R233E/E240N 351Y155F/R338E/T343R/R403E/E410N Y[155]F/R170E/T175R/R233E/E240N 352Y155F/K247N/N249S/R318Y/R338E/ Y[155]F/K82N/N84S/R150Y/R170E/ 353T343R/R403E/E410N T175R/R233E/E240N K247N/N249S/R318Y/R338E/T343R/K82N/N84S/R150Y/R170E/T175R/ 354 R403E/E410N R233E/E240NK228N/I251S/R318Y/R338E/R403E/ K63N/I86S/R150Y/R170E/R233E/ 355 E410NE240N Y155F/K228N/I251S/R318Y/R338E/ Y[155]F/K63N/I86S/R150Y/R170E/ 356R403E/E410N R233E/E240N N260S/R318Y/R338E/T343R/R403E/N95S/R150Y/R170E/T175R/R233E/ 357 E410N E240NY155F/N260S/R318Y/R338E/T343R/ Y[155]F/N95S/R150Y/R170E/T175R/ 358R403E/E410N R233E/E240N K228N/K247N/N249S/R318Y/R338E/K63N/K82N/N84S/R150Y/R170E/ 359 T343R/R403E/E410N T175R/R233E/E240NY155F/K228N/K247N/N249S/R318Y/ Y[155]F/K63N/K82N/N84S/R150Y/ 360R338E/T343R/R403E/E410N R170E/T175R/R233E/E240N Y155F/R338E/T343R/R403EY[155]F/R170E/T175R/R233E 361 R338E/T343R/R403E R170E/T175R/R233E 362Y155F/R338E/T343R/R403E/E410S Y[155]F/R170E/T175R/R233E/E240S 363Y155F/N260S/R338E/T343R/R403E Y[155]F/N95S/R170E/T175R/R233E 364Y155F/I251S/R338E/T343R/R403E Y[155]F/I86S/R170E/T175R/R233E 365R318Y/R338E/T343R/R403E/E410S R150Y/R170E/T175R/R233E/E240S 366Y155F/K247N/N249S/T343R/R403E Y[155]F/K82N/N84S/T175R/R233E 367Y155F/K247N/N249S/R318Y/R338E/ Y[155]F/K82N/N84S/R150Y/R170E/ 368T343R/R403E T175R/R233E K247N/N249S/R318Y/R338E/T343R/K82N/N84S/R150Y/R170E/T175R/ 369 R403E R233EY155F/K247N/N249S/R338E/T343R/ Y[155]F/K82N/N84S/R170E/T175R/ 370R403E/E410N R233E/E240N K247N/N249S/R338E/T343R/R403E/K82N/N84S/R170E/T175R/R233E/ 371 E410N E240NY155F/K247N/N249S/R318Y/R338E Y[155]F/K82N/N84S/R150Y/R170E 372Y155F/K247N/N249S/R318Y/T343R Y[155]F/K82N/N84S/R150Y/T175R 373Y155F/K247N/N249S/R318Y/R403E Y[155]F/K82N/N84S/R150Y/R233E 374Y155F/K247N/N249S/R318Y/E410N Y[155]F/K82N/N84S/R150Y/E240N 375Y155F/K247N/N249S/R338E/R403E Y[155]F/K82N/N84S/R170E/R233E 376Y155F/K247N/N249S/R338E/T343R Y[155]F/K82N/N84S/R170E/T175R 377Y155F/K247N/N249S/R318Y/R338E/ Y[155]F/K82N/N84S/R150Y/R170E/ 378T343R/E410N T175R/E240N K247N/N249S/R318Y/R338E/T343R/K82N/N84S/R150Y/R170E/T175R/ 379 E410N E240NY155F/K247N/N249S/R318Y/T343R/ Y[155]F/K82N/N84S/R150Y/T175R/ 380R403E/E410N R233E/E240N K247N/N249S/R318Y/T343R/R403E/K82N/N84S/R150Y/T175R/R233E/ 381 E410N E240NY155F/K247N/N249S/R338E/E410N Y[155]F/K82N/N84S/R170E/E240N 382Y155F/K247N/N249S/R318Y/T343R/ Y[155]F/K82N/N84S/R150Y/T175R/ 383 R403ER233E K247N/N249S/R318Y/T343R/R403E K82N/N84S/R150Y/T175R/R233E 384Y155F/K247N/N249S/R318Y/T343R/ Y[155]F/K82N/N84S/R150Y/T175R/ 385 E410NE240N K247N/N249S/R318Y/T343R/E410N K82N/N84S/R150Y/T175R/E240N 386Y155F/K247N/N249S/R338E/T343R/ Y[155]F/K82N/N84S/R170E/T175R/ 387 R403ER233E K247N/N249S/R338E/T343R/R403E K82N/N84S/R170E/T175R/R233E 388Y155F/K247N/N249S/R338E/T343R/ Y[155]F/K82N/N84S/R170E/T175R/ 389 E410NE240N K247N/N249S/R338E/T343R/E410N K82N/N84S/R170E/T175R/E240N 390Y155F/K247N/N249S/T343R/R403E/ Y[155]F/K82N/N84S/T175R/R233E/ 391 E410NE240N K247N/N249S/T343R/R403E/E410N K82N/N84S/T175R/R233E/E240N 392Y155F/R318Y/R338E/T343R Y[155]F/R150Y/R170E/T175R 393 R318Y/R338E/T343RR150Y/R170E/T175R 394 Y155F/R318Y/T343R/R403E Y[155]F/R150Y/T175R/R233E395 Y155F/T343R/R403E/E410N Y[155]F/T175R/R233E/E240N 396Y155F/K247N/N249S/R318Y/R338E/ Y[155]F/K82N/N84S/R150Y/R170E/ 397 T343RT175R K247N/N249S/R318Y/R338E/T343R K82N/N84S/R150Y/R170E/T175R 398Y155F/K247N/N249S/T343R/E410N Y[155]F/K82N/N84S/T175R/E240N 399Y155F/K247N/N249S/R403E/E410N Y[155]F/K82N/N84S/R233E/E240N 400Y155F/R338E/T343R/E410N Y[155]F/R170E/T175R/E240N 401 R338E/T343R/E410NR170E/T175R/E240N 402 Y155F/R318Y/T343R/E410N Y[155]F/R150Y/T175R/E240N403 R318Y/T343R/E410N R150Y/T175R/E240N 404K228N/R318Y/R338E/T343R/R403E/ K63N/R150Y/R170E/T175R/R233E/ 405 E410NE240N K228N/K247N/N249S/R318Y/R338E/ K63N/K82N/N84S/R150Y/R170E/ 406T343R/R403E T175R/R233E K228N/K247N/N249S/R318Y/R338E/K63N/K82N/N84S/R150Y/R170E/ 407 T343R/E410N T175R/E240NK228N/K247N/N249S/R318Y/T343R/ K63N/K82N/N84S/R150Y/T175R/ 408R403E/E410N R233E/E240N Y155F/R338E/R403E/E410NY[155]F/R170E/R233E/E240N 409 Y155F/R318Y/R338E/R403EY[155]F/R150Y/R170E/R233E 410 Y155F/R318Y/R403E/E410NY[155]F/R150Y/R233E/E240N 411

3. Conjugates and Fusion Proteins

The modified FIX polypeptides provided herein can be conjugated or fusedto another polypeptide or other moiety, such as a polymer. In someinstances, the conjugation or fusion is effected to increase serumhalf-life. Exemplary polypeptides to which the modified FIX polypeptidesprovided herein can be fused include, but are not limited to, serumalbumin, Fc, FcRn and tranferrin (see, e.g., Sheffield, W. P. et al.,(2004) Br. J. Haematol. 126(4):565-73; U.S. Patent Publication No.20050147618; International Patent Publication Nos. WO2007112005 andWO2004101740).

The modified FIX polypeptides provided herein can be conjugated to apolymer, such as dextran, a polyethylene glycol (pegylation(PEG)) orsialyl moiety, or other such polymers, such as natural or sugarpolymers. In one example, the polypeptides are conjugated to dextrans,such as described elsewhere (Zambaux et al., (1998) J. Protein Chem.17(3):279-84). Various methods of modifying polypeptides by covalentlyattaching (conjugating) a PEG or PEG derivative (i.e. “PEGylation”) areknown in the art (see e.g., US20060104968, U.S. Pat. No. 5,672,662, U.S.Pat. No. 6,737,505 and US 20040235734). Techniques for PEGylationinclude, but are not limited to, specialized linkers and couplingchemistries (see e.g., Harris, Adv. Drug Deliv. Rev. 54:459-476, 2002),attachment of multiple PEG moieties to a single conjugation site (suchas via use of branched PEGs; see e.g., Veronese et al., Bioorg. Med.Chem. Lett. 12:177-180, 2002), site-specific PEGylation and/ormono-PEGylation (see e.g., Chapman et al., Nature Biotech. 17:780-783,1999), site-directed enzymatic PEGylation (see e.g., Sato, Adv. DrugDeliv. Rev., 54:487-504, 2002), and glycoPEGylation (U.S. PatentPublication Nos. 20080050772, 20080146494, 20080050772, 20080187955 and20080206808). Methods and techniques described in the art can produceproteins having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more PEG or PEGderivatives attached to a single protein molecule (see e.g., U.S.2006/0104968). Thus, the modified FIX polypeptide provided herein can bepegylated, including glycopegylated, using any method known in the art,such as any described in U.S. Pat. Nos. 5,969,040, 5,621,039, 6,423,826,U.S. Patent Publication Nos. 20030211094, 20070254840, 20080188414,2008000422, 20080050772, 20080146494, 20080050772, 20080187955 and20080206808, International Patent Publication Nos. WO2007112005,WO2007135182, WO2008082613, WO2008119815, WO2008119815.

In some instances, the modified FIX polypeptides include amino acidreplacements to facilitate conjugation to another moiety. For example,cysteine residues can be incorporated into the FIX polypeptide tofacilitate conjugation to polymers. Exemplary amino acid replacementmodifications for this purpose include, but are not limited to, D47C,Q50C, S53C, L57C, I66C, N67C, S68C, E70C, W72C, P74C, K80C, L84C, V86C,N89C, 190C, K91C, R94C, K100C, N101C, S102C, A103C, D104C, N105C, K106C,V108C, E114C, R116C, E119C, N120C, Q121C, S123C, E125C, P129C, S138C,T140C, S141C, K142C, A146C, E147C, E162C, T163C, I164C, L165C, D166C,N167C, I168C, T169C, Q170C, S171C, T172C, Q173C, S174C, F175C, N176C,D177C, F178C, T179C, R180C, E185C, D186C, K188C, P189C, K201C, V202C,D203C, E224C, T225C, K228C, E239C, E240C, T241C, H243C, K247C, N249C,R252C, H257C, N260C, A261C, A262C, I263C, K265C, E277C, F314C, R318C,L321C, K341C, E372C, E374C, M391C, K392C, N406C, K413C and T415C(corresponding to a mature FIX polypeptide set forth in SEQ ID NO:3).

E. PRODUCTION OF FIX POLYPEPTIDES

FIX polypeptides, including modified FIX polypeptides, or domainsthereof, of FIX can be obtained by methods well known in the art forprotein purification and recombinant protein expression. Any methodknown to those of skill in the art for identification of nucleic acidsthat encode desired genes can be used. Any method available in the artcan be used to obtain a full length (i.e., encompassing the entirecoding region) cDNA or genomic DNA clone encoding a FIX polypeptide orother vitamin-K polypeptide, such as from a cell or tissue source, suchas for example from liver. Modified FIX polypeptides can be engineeredas described herein, such as by site-directed mutagenesis.

FIX can be cloned or isolated using any available methods known in theart for cloning and isolating nucleic acid molecules. Such methodsinclude PCR amplification of nucleic acids and screening of libraries,including nucleic acid hybridization screening, antibody-based screeningand activity-based screening.

Methods for amplification of nucleic acids can be used to isolatenucleic acid molecules encoding a FIX polypeptide, including forexample, polymerase chain reaction (PCR) methods. A nucleic acidcontaining material can be used as a starting material from which aFIX-encoding nucleic acid molecule can be isolated. For example, DNA andmRNA preparations, cell extracts, tissue extracts (e.g. from liver),fluid samples (e.g. blood, serum, saliva), samples from healthy and/ordiseased subjects can be used in amplification methods. Nucleic acidlibraries also can be used as a source of starting material. Primers canbe designed to amplify a FIX-encoding molecule. For example, primers canbe designed based on expressed sequences from which a FIX is generated.Primers can be designed based on back-translation of a FIX amino acidsequence. Nucleic acid molecules generated by amplification can besequenced and confirmed to encode a FIX polypeptide.

Additional nucleotide sequences can be joined to a FIX-encoding nucleicacid molecule, including linker sequences containing restrictionendonuclease sites for the purpose of cloning the synthetic gene into avector, for example, a protein expression vector or a vector designedfor the amplification of the core protein coding DNA sequences.Furthermore, additional nucleotide sequences specifying functional DNAelements can be operatively linked to a FIX-encoding nucleic acidmolecule. Examples of such sequences include, but are not limited to,promoter sequences designed to facilitate intracellular proteinexpression, and secretion sequences designed to facilitate proteinsecretion. Additional nucleotide sequences such as sequences specifyingprotein binding regions also can be linked to FIX-encoding nucleic acidmolecules. Such regions include, but are not limited to, sequences tofacilitate uptake of FIX into specific target cells, or otherwiseenhance the pharmacokinetics of the synthetic gene.

The identified and isolated nucleic acids can then be inserted into anappropriate cloning vector. A large number of vector-host systems knownin the art can be used. Possible vectors include, but are not limitedto, plasmids or modified viruses, but the vector system must becompatible with the host cell used. Such vectors include, but are notlimited to, bacteriophages such as lambda derivatives, or plasmids suchas pBR322 or pUC plasmid derivatives or the Bluescript vector(Stratagene, La Jolla, Calif.). The insertion into a cloning vector can,for example, be accomplished by ligating the DNA fragment into a cloningvector which has complementary cohesive termini. Insertion can beeffected using TOPO cloning vectors (Invitrogen, Carlsbad, Calif.). Ifthe complementary restriction sites used to fragment the DNA are notpresent in the cloning vector, the ends of the DNA molecules can beenzymatically modified. Alternatively, any site desired can be producedby ligating nucleotide sequences (linkers) onto the DNA termini; theseligated linkers can contain specific chemically synthesizedoligonucleotides encoding restriction endonuclease recognitionsequences. In an alternative method, the cleaved vector and FIX proteingene can be modified by homopolymeric tailing. Recombinant molecules canbe introduced into host cells via, for example, transformation,transfection, infection, electroporation and sonoporation, so that manycopies of the gene sequence are generated.

In specific embodiments, transformation of host cells with recombinantDNA molecules that incorporate the isolated FIX protein gene, cDNA, orsynthesized DNA sequence enables generation of multiple copies of thegene. Thus, the gene can be obtained in large quantities by growingtransformants, isolating the recombinant DNA molecules from thetransformants and, when necessary, retrieving the inserted gene from theisolated recombinant DNA.

1. Vectors and Cells

For recombinant expression of one or more of the FIX proteins, thenucleic acid containing all or a portion of the nucleotide sequenceencoding the FIX protein can be inserted into an appropriate expressionvector, i.e., a vector that contains the necessary elements for thetranscription and translation of the inserted protein coding sequence.Exemplary of such a vector is any mammalian expression vector such as,for example, pCMV. The necessary transcriptional and translationalsignals also can be supplied by the native promoter for a FIX genes,and/or their flanking regions.

Also provided are vectors that contain nucleic acid encoding the FIX ormodified FIX. Cells containing the vectors also are provided. The cellsinclude eukaryotic and prokaryotic cells, and the vectors are anysuitable for use therein.

Prokaryotic and eukaryotic cells, including endothelial cells,containing the vectors are provided. Such cells include bacterial cells,yeast cells, fungal cells, Archea, plant cells, insect cells and animalcells. The cells are used to produce a FIX polypeptide or modified FIXpolypeptide thereof by growing the above-described cells underconditions whereby the encoded FIX protein is expressed by the cell, andrecovering the expressed FIX protein. For purposes herein, the FIX canbe secreted into the medium.

In one embodiment, vectors containing a sequence of nucleotides thatencodes a polypeptide that has FIX activity and contains all or aportion of the FIX polypeptide, or multiple copies thereof, areprovided. The vectors can be selected for expression of the FIXpolypeptide or modified FIX polypeptide thereof in the cell or such thatthe FIX protein is expressed as a secreted protein. When the FIX isexpressed the nucleic acid is linked to nucleic acid encoding asecretion signal, such as the Saccharomyces cerevisiae α-mating factorsignal sequence or a portion thereof, or the native signal sequence.

A variety of host-vector systems can be used to express the proteincoding sequence. These include but are not limited to mammalian cellsystems infected with virus (e.g. vaccinia virus, adenovirus and otherviruses); insect cell systems infected with virus (e.g. baculovirus);microorganisms such as yeast containing yeast vectors; or bacteriatransformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA. Theexpression elements of vectors vary in their strengths andspecificities. Depending on the host-vector system used, any one of anumber of suitable transcription and translation elements can be used.

Any methods known to those of skill in the art for the insertion of DNAfragments into a vector can be used to construct expression vectorscontaining a chimeric gene containing appropriatetranscriptional/translational control signals and protein codingsequences. These methods can include in vitro recombinant DNA andsynthetic techniques and in vivo recombinants (genetic recombination).Expression of nucleic acid sequences encoding a FIX polypeptide ormodified FIX polypeptide, or domains, derivatives, fragments or homologsthereof, can be regulated by a second nucleic acid sequence so that thegenes or fragments thereof are expressed in a host transformed with therecombinant DNA molecule(s). For example, expression of the proteins canbe controlled by any promoter/enhancer known in the art. In a specificembodiment, the promoter is not native to the genes for a FIX protein.Promoters which can be used include but are not limited to the SV40early promoter (Bernoist and Chambon, Nature 290:304-310 (1981)), thepromoter contained in the 3′ long terminal repeat of Rous sarcoma virus(Yamamoto et al. Cell 22:787-797 (1980)), the herpes thymidine kinasepromoter (Wagner et al., Proc. Natl. Acad. Sci. USA 78:1441-1445(1981)), the regulatory sequences of the metallothionein gene (Brinsteret al., Nature 296:39-42 (1982)); prokaryotic expression vectors such asthe β-lactamase promoter (Jay et al., (1981) Proc. Natl. Acad. Sci. USA78:5543) or the tac promoter (DeBoer et al., Proc. Natl. Acad. Sci. USA80:21-25 (1983)); see also “Useful Proteins from Recombinant Bacteria”:in Scientific American 242:79-94 (1980)); plant expression vectorscontaining the nopaline synthetase promoter (Herrara-Estrella et al.,Nature 303:209-213 (1984)) or the cauliflower mosaic virus 35S RNApromoter (Garder et al., Nucleic Acids Res. 9:2871 (1981)), and thepromoter of the photosynthetic enzyme ribulose bisphosphate carboxylase(Herrera-Estrella et al., Nature 310:115-120 (1984)); promoter elementsfrom yeast and other fungi such as the Gal4 promoter, the alcoholdehydrogenase promoter, the phosphoglycerol kinase promoter, thealkaline phosphatase promoter, and the following animal transcriptionalcontrol regions that exhibit tissue specificity and have been used intransgenic animals: elastase I gene control region which is active inpancreatic acinar cells (Swift et al., Cell 38:639-646 (1984); Ornitz etal., Cold Spring Harbor Symp. Quant. Biol. 50:399-409 (1986); MacDonald,Hepatology 7:425-515 (1987)); insulin gene control region which isactive in pancreatic beta cells (Hanahan et al., Nature 315:115-122(1985)), immunoglobulin gene control region which is active in lymphoidcells (Grosschedl et al., Cell 38:647-658 (1984); Adams et al., Nature318:533-538 (1985); Alexander et al., Mol. Cell Biol. 7:1436-1444(1987)), mouse mammary tumor virus control region which is active intesticular, breast, lymphoid and mast cells (Leder et al., Cell45:485-495 (1986)), albumin gene control region which is active in liver(Pinckert et al., Genes and Devel. 1:268-276 (1987)), alpha-fetoproteingene control region which is active in liver (Krumlauf et al., Mol.Cell. Biol. 5:1639-1648 (1985); Hammer et al., Science 235:53-58 1987)),alpha-1 antitrypsin gene control region which is active in liver (Kelseyet al., Genes and Devel. 1:161-171 (1987)), beta globin gene controlregion which is active in myeloid cells (Magram et al., Nature315:338-340 (1985); Kollias et al., Cell 46:89-94 (1986)), myelin basicprotein gene control region which is active in oligodendrocyte cells ofthe brain (Readhead et al., Cell 48:703-712 (1987)), myosin lightchain-2 gene control region which is active in skeletal muscle (Shani,Nature 314:283-286 (1985)), and gonadotrophic releasing hormone genecontrol region which is active in gonadotrophs of the hypothalamus(Mason et al., Science 234:1372-1378 (1986)).

In a specific embodiment, a vector is used that contains a promoteroperably linked to nucleic acids encoding a FIX polypeptide or modifiedFIX polypeptide, or a domain, fragment, derivative or homolog, thereof,one or more origins of replication, and optionally, one or moreselectable markers (e.g., an antibiotic resistance gene). Vectors andsystems for expression of FIX polypeptides include the well known Pichiavectors (available, for example, from Invitrogen, San Diego, Calif.),particularly those designed for secretion of the encoded proteins.Exemplary plasmid vectors for expression in mammalian cells include, forexample, pCMV. Exemplary plasmid vectors for transformation of E. colicells, include, for example, the pQE expression vectors (available fromQiagen, Valencia, Calif.; see also literature published by Qiagendescribing the system). pQE vectors have a phage T5 promoter (recognizedby E. coli RNA polymerase) and a double lac operator repression moduleto provide tightly regulated, high-level expression of recombinantproteins in E. coli, a synthetic ribosomal binding site (RBS II) forefficient translation, a 6×His tag coding sequence, t₀ and T1transcriptional terminators, ColE1 origin of replication, and abeta-lactamase gene for conferring ampicillin resistance. The pQEvectors enable placement of a 6×His tag at either the N- or C-terminusof the recombinant protein. Such plasmids include pQE 32, pQE 30, andpQE 31 which provide multiple cloning sites for all three reading framesand provide for the expression of N-terminally 6×His-tagged proteins.Other exemplary plasmid vectors for transformation of E. coli cells,include, for example, the pET expression vectors (see, U.S. Pat. No.4,952,496; available from NOVAGEN, Madison, Wis.; see, also literaturepublished by Novagen describing the system). Such plasmids include pET11a, which contains the T7lac promoter, T7 terminator, the inducible E.coli lac operator, and the lac repressor gene; pET 12a-c, which containsthe T7 promoter, T7 terminator, and the E. coli ompT secretion signal;and pET 15b and pET19b (NOVAGEN, Madison, Wis.), which contain aHis-Tag™ leader sequence for use in purification with a His column and athrombin cleavage site that permits cleavage following purification overthe column, the T7-lac promoter region and the T7 terminator.

2. Expression Systems

FIX polypeptides (modified and unmodified) can be produced by anymethods known in the art for protein production including in vitro andin vivo methods such as, for example, the introduction of nucleic acidmolecules encoding FIX into a host cell, host animal and expression fromnucleic acid molecules encoding FIX in vitro. FIX and modified FIXpolypeptides can be expressed in any organism suitable to produce therequired amounts and forms of a FIX polypeptide needed foradministration and treatment. Expression hosts include prokaryotic andeukaryotic organisms such as E. coli, yeast, plants, insect cells,mammalian cells, including human cell lines and transgenic animals.Expression hosts can differ in their protein production levels as wellas the types of post-translational modifications that are present on theexpressed proteins. The choice of expression host can be made based onthese and other factors, such as regulatory and safety considerations,production costs and the need and methods for purification.

Expression in eukaryotic hosts can include expression in yeasts such asSaccharomyces cerevisiae and Pichia pastoris, insect cells such asDrosophila cells and lepidopteran cells, plants and plant cells such astobacco, corn, rice, algae, and lemna. Eukaryotic cells for expressionalso include mammalian cells lines such as Chinese hamster ovary (CHO)cells or baby hamster kidney (BHK) cells. Eukaryotic expression hostsalso include production in transgenic animals, for example, includingproduction in serum, milk and eggs. Transgenic animals for theproduction of wild-type FIX polypeptides are known in the art (U.S.Patent Publication Nos. 2002-0166130 and 2004-0133930) and can beadapted for production of modified FIX polypeptides provided herein.

Many expression vectors are available and known to those of skill in theart for the expression of FIX. The choice of expression vector isinfluenced by the choice of host expression system. Such selection iswell within the level of skill of the skilled artisan. In general,expression vectors can include transcriptional promoters and optionallyenhancers, translational signals, and transcriptional and translationaltermination signals. Expression vectors that are used for stabletransformation typically have a selectable marker which allows selectionand maintenance of the transformed cells. In some cases, an origin ofreplication can be used to amplify the copy number of the vectors in thecells.

FIX or modified FIX polypeptides also can be utilized or expressed asprotein fusions. For example, a fusion can be generated to addadditional functionality to a polypeptide. Examples of fusion proteinsinclude, but are not limited to, fusions of a signal sequence, a tagsuch as for localization, e.g. a his₆ tag or a myc tag, or a tag forpurification, for example, a GST fusion, and a sequence for directingprotein secretion and/or membrane association.

In one embodiment, the FIX polypeptide or modified FIX polypeptides canbe expressed in an active form, whereby activation is achieved byincubation of the polypeptide activated factor XI (FXIa) followingsecretion. In another embodiment, the protease is expressed in aninactive, zymogen form.

Methods of production of FIX polypeptides can include coexpression ofone or more additional heterologous polypeptides that can aid in thegeneration of the FIX polypeptides. For example, such polypeptides cancontribute to the post-translation processing of the FIX polypeptides.Exemplary polypeptides include, but are not limited to, peptidases thathelp cleave FIX precursor sequences, such as the propeptide sequence,and enzymes that participate in the modification of the FIX polypeptide,such as by glycosylation, hydroxylation, carboxylation, orphosphorylation, for example. An exemplary peptidase that can becoexpressed with FIX is PACE/furin (or PACE-SOL), which aids in thecleavage of the FIX propeptide sequence. An exemplary protein that aidsin the carboxylation of the FIX polypeptide is the warfarin-sensitiveenzyme vitamin K 2,3-epoxide reductase (VKOR), which produces reducedvitamin K for utilization as a cofactor by the vitamin K-dependentγ-carboxylase (Wajih et al., J. Biol. Chem. 280(36)31603-31607). Asubunit of this enzyme, VKORC1, can be coexpressed with the modified FIXpolypeptide to increase the γ-carboxylation The one or more additionalpolypeptides can be expressed from the same expression vector as the FIXpolypeptide or from a different vector.

a. Prokaryotic Expression

Prokaryotes, especially E. coli, provide a system for producing largeamounts of FIX (see, for example, Platis et al. (2003) Protein Exp.Purif. 31(2): 222-30; and Khalilzadeh et al. (2004) J. Ind. Microbiol.Biotechnol. 31(2): 63-69). Transformation of E. coli is a simple andrapid technique well known to those of skill in the art. Expressionvectors for E. coli can contain inducible promoters that are useful forinducing high levels of protein expression and for expressing proteinsthat exhibit some toxicity to the host cells. Examples of induciblepromoters include the lac promoter, the trp promoter, the hybrid tacpromoter, the T7 and SP6 RNA promoters and the temperature regulatedλP_(L) promoter.

FIX can be expressed in the cytoplasmic environment of E. coli. Thecytoplasm is a reducing environment and for some molecules, this canresult in the formation of insoluble inclusion bodies. Reducing agentssuch as dithiothreitol and β-mercaptoethanol and denaturants (e.g., suchas guanidine-HCl and urea) can be used to resolubilize the proteins. Analternative approach is the expression of FIX in the periplasmic spaceof bacteria which provides an oxidizing environment and chaperonin-likeand disulfide isomerases leading to the production of soluble protein.Typically, a leader sequence is fused to the protein to be expressedwhich directs the protein to the periplasm. The leader is then removedby signal peptidases inside the periplasm. Examples ofperiplasmic-targeting leader sequences include the pelB leader from thepectate lyase gene and the leader derived from the alkaline phosphatasegene. In some cases, periplasmic expression allows leakage of theexpressed protein into the culture medium. The secretion of proteinsallows quick and simple purification from the culture supernatant.Proteins that are not secreted can be obtained from the periplasm byosmotic lysis. Similar to cytoplasmic expression, in some cases proteinscan become insoluble and denaturants and reducing agents can be used tofacilitate solubilization and refolding. Temperature of induction andgrowth also can influence expression levels and solubility. Typically,temperatures between 25° C. and 37° C. are used. Mutations also can beused to increase solubility of expressed proteins. Typically, bacteriaproduce aglycosylated proteins. Thus, for the production of thehyperglycosylated FIX polypeptides provided herein, glycosylation can beadded in vitro after purification from host cells.

b. Yeast

Yeasts such as Saccharomyces cerevisiae, Schizosaccharomyces pombe,Yarrowia lipolytica, Kluyveromyces lactis, and Pichia pastoris areuseful expression hosts for FIX (see for example, Skoko et al. (2003)Biotechnol. Appl. Biochem. 38(Pt3):257-65). Yeast can be transformedwith episomal replicating vectors or by stable chromosomal integrationby homologous recombination. Typically, inducible promoters are used toregulate gene expression. Examples of such promoters include GAL1, GAL7,and GAL5 and metallothionein promoters such as CUP1. Expression vectorsoften include a selectable marker such as LEU2, TRP1, HIS3, and URA3 forselection and maintenance of the transformed DNA. Proteins expressed inyeast are often soluble and co-expression with chaperonins, such as Bipand protein disulfide isomerase, can improve expression levels andsolubility. Additionally, proteins expressed in yeast can be directedfor secretion using secretion signal peptide fusions such as the yeastmating type alpha-factor secretion signal from Saccharomyces cerevisiaeand fusions with yeast cell surface proteins such as the Aga2p matingadhesion receptor or the Arxula adeninivorans glucoamylase. A proteasecleavage site (e.g., the Kex-2 protease) can be engineered to remove thefused sequences from the polypeptides as they exit the secretionpathway. Yeast also is capable of glycosylation at Asn-X-Ser/Thr motifs.

c. Insects and Insect Cells

Insects and insect cells, particularly using a baculovirus expressionsystem, are useful for expressing polypeptides such as FIX or modifiedforms thereof (see, for example, Muneta et al. (2003) J. Vet. Med. Sci.65(2):219-23). Insect cells and insect larvae, including expression inthe haemolymph, express high levels of protein and are capable of mostof the post-translational modifications used by higher eukaryotes.Baculoviruses have a restrictive host range which improves the safetyand reduces regulatory concerns of eukaryotic expression. Typically,expression vectors use a promoter such as the polyhedrin promoter ofbaculovirus for high level expression. Commonly used baculovirus systemsinclude baculoviruses such as Autographa californica nuclearpolyhedrosis virus (AcNPV), and the Bombyx mori nuclear polyhedrosisvirus (BmNPV) and an insect cell line such as Sf9 derived fromSpodoptera frugiperda, Pseudaletia unipuncta (A7S) and Danaus plexippus(DpN1). For high level expression, the nucleotide sequence of themolecule to be expressed is fused immediately downstream of thepolyhedrin initiation codon of the virus. Mammalian secretion signalsare accurately processed in insect cells and can be used to secrete theexpressed protein into the culture medium. In addition, the cell linesPseudaletia unipuncta (A7S) and Danaus plexippus (DpN1) produce proteinswith glycosylation patterns similar to mammalian cell systems.

An alternative expression system in insect cells is the use of stablytransformed cells. Cell lines such as the Schnieder 2 (S2) and Kc cells(Drosophila melanogaster) and C7 cells (Aedes albopictus) can be usedfor expression. The Drosophila metallothionein promoter can be used toinduce high levels of expression in the presence of heavy metalinduction with cadmium or copper. Expression vectors are typicallymaintained by the use of selectable markers such as neomycin andhygromycin.

d. Mammalian Cells

Mammalian expression systems can be used to express FIX polypeptides.Expression constructs can be transferred to mammalian cells by viralinfection such as adenovirus or by direct DNA transfer such asliposomes, calcium phosphate, DEAE-dextran and by physical means such aselectroporation and microinjection. Expression vectors for mammaliancells typically include an mRNA cap site, a TATA box, a translationalinitiation sequence (Kozak consensus sequence) and polyadenylationelements. Such vectors often include transcriptional promoter-enhancersfor high level expression, for example the SV40 promoter-enhancer, thehuman cytomegalovirus (CMV) promoter, and the long terminal repeat ofRous sarcoma virus (RSV). These promoter-enhancers are active in manycell types. Tissue and cell-type promoters and enhancer regions also canbe used for expression. Exemplary promoter/enhancer regions include, butare not limited to, those from genes such as elastase I, insulin,immunoglobulin, mouse mammary tumor virus, albumin, alpha-fetoprotein,alpha 1-antitrypsin, beta-globin, myelin basic protein, myosin lightchain-2, and gonadotropic releasing hormone gene control. Selectablemarkers can be used to select for and maintain cells with the expressionconstruct. Examples of selectable marker genes include, but are notlimited to, hygromycin B phosphotransferase, adenosine deaminase,xanthine-guanine phosphoribosyl transferase, aminoglycosidephosphotransferase, dihydrofolate reductase and thymidine kinase. Fusionwith cell surface signaling molecules such as TCR-ζ and Fc_(ε)RI-γ candirect expression of the proteins in an active state on the cellsurface.

Many cell lines are available for mammalian expression including mouse,rat human, monkey, and chicken and hamster cells. Exemplary cell linesinclude, but are not limited to, BHK (i.e. BHK-21 cells), 293-F, CHO,CHO Express (CHOX; Excellgene), Balb/3T3, HeLa, MT2, mouse NSO(non-secreting) and other myeloma cell lines, hybridoma andheterohybridoma cell lines, lymphocytes, fibroblasts, Sp2/0, COS,NIH3T3, HEK293, 293S, 293T, 2B8, and HKB cells. Cell lines also areavailable adapted to serum-free media which facilitates purification ofsecreted proteins from the cell culture media. One such example is theserum free EBNA-1 cell line (Pham et al., (2003) Biotechnol. Bioeng.84:332-42). Expression of recombinant FIX polypeptides exhibitingsimilar structure and post-translational modifications as plasma-derivedFIX are known in the art. Methods of optimizing vitamin K-dependentprotein expression are known. For example, supplementation of vitamin Kin culture medium or co-expression of vitamin K-dependent γ-carboxylases(Wajih et al., J. Biol. Chem. 280(36)31603-31607) can aid inpost-translational modification of vitamin K-dependent proteins, such asFIX polypeptides.

e. Plants

Transgenic plant cells and plants can be used for the expression of FIX.Expression constructs are typically transferred to plants using directDNA transfer such as microprojectile bombardment and PEG-mediatedtransfer into protoplasts, and with agrobacterium-mediatedtransformation. Expression vectors can include promoter and enhancersequences, transcriptional termination elements, and translationalcontrol elements. Expression vectors and transformation techniques areusually divided between dicot hosts, such as Arabidopsis and tobacco,and monocot hosts, such as corn and rice. Examples of plant promotersused for expression include the cauliflower mosaic virus promoter, thenopaline synthase promoter, the ribose bisphosphate carboxylase promoterand the ubiquitin and UBQ3 promoters. Selectable markers such ashygromycin, phosphomannose isomerase and neomycin phosphotransferase areoften used to facilitate selection and maintenance of transformed cells.Transformed plant cells can be maintained in culture as cells,aggregates (callus tissue) or regenerated into whole plants. Becauseplants have different glycosylation patterns than mammalian cells, thiscan influence the choice to produce FIX in these hosts. Transgenic plantcells also can include algae engineered to produce proteins (see, forexample, Mayfield et al. (2003) Proc Natl Acad Sci USA 100:438-442).Because plants have different glycosylation patterns than mammaliancells, this can influence the choice to produce FIX in these hosts.

2. Purification

Methods for purification of FIX polypeptides from host cells depend onthe chosen host cells and expression systems. For secreted molecules,proteins are generally purified from the culture media after removingthe cells. For intracellular expression, cells can be lysed and theproteins purified from the extract. When transgenic organisms such astransgenic plants and animals are used for expression, tissues or organscan be used as starting material to make a lysed cell extract.Additionally, transgenic animal production can include the production ofpolypeptides in milk or eggs, which can be collected, and if necessaryfurther the proteins can be extracted and further purified usingstandard methods in the art.

FIX can be purified using standard protein purification techniques knownin the art including but not limited to, SDS-PAGE, size fraction andsize exclusion chromatography, ammonium sulfate precipitation, chelatechromatography and ionic exchange chromatography. For example, FIXpolypeptides can be purified by anion exchange chromatography, such asdescribed in Example 1, below. Exemplary of a method to purify FIXpolypeptides is by using an ion exchange column that permits binding ofany polypeptide that has a functional Gla domain, followed by elution inthe presence of calcium. Affinity purification techniques also can beused to improve the efficiency and purity of the preparations. Forexample, antibodies, receptors and other molecules that bind FIX can beused in affinity purification. Expression constructs also can beengineered to add an affinity tag such as a myc epitope, GST fusion orHis₆ and affinity purified with myc antibody, glutathione resin, andNi-resin, respectively, to a protein. Purity can be assessed by anymethod known in the art including gel electrophoresis and staining andspectrophotometric techniques.

The FIX polypeptide can be expressed and purified to be in an inactiveform (zymogen form) or alternatively the expressed protease can bepurified into an active form, such as by autocatalysis. For example, FIXpolypeptides that have been activated via proteolytic cleavage afterR145 and R180 can be prepared in vitro (i.e. FIXa; two-chain form). TheFIX polypeptides can be first prepared by any of the methods ofproduction described herein, including, but not limited to, productionin mammalian cells followed by purification. Cleavage of the FIXpolypeptides into the active protease form, FIXa, can be accomplished byincubation with factor XIa. In some examples, this is performed in thepresence of calcium and phospholipids.

3. Fusion Proteins

Fusion proteins containing a modified FIX polypeptide and one or moreother polypeptides also are provided. Pharmaceutical compositionscontaining such fusion proteins formulated for administration by asuitable route are provided. Fusion proteins are formed by linking inany order the modified FIX polypeptide and an agent, such as an antibodyor fragment thereof, growth factor, receptor, ligand, and other suchagent for the purposes of facilitating the purification of a FIXpolypeptide, altering the pharmacodynamic properties of a FIXpolypeptide by directing, for example, by directing the polypeptide to atargeted cell or tissue, and/or increasing the expression or secretionof the FIX polypeptide. Typically any FIX fusion protein retains atleast about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% coagulant activitycompared with a non-fusion FIX polypeptide, including 96%, 97%, 98%, 99%or greater coagulant activity compared with a non-fusion polypeptide.

Linkage of a FIX polypeptide with another polypeptide can be effecteddirectly or indirectly via a linker. In one example, linkage can be bychemical linkage, such as via heterobifunctional agents or thiollinkages or other such linkages. Fusion also can be effected byrecombinant means. Fusion of a FIX polypeptide to another polypeptidecan be to the N- or C-terminus of the FIX polypeptide. Non-limitingexamples of polypeptides that can be used in fusion proteins with a FIXpolypeptide provided herein include, for example, a GST (glutathioneS-transferase) polypeptide, Fc domain from immunoglobulin G, albumin, ora heterologous signal sequence. The fusion proteins can containadditional components, such as E. coli maltose binding protein (MBP)that aid in uptake of the protein by cells (see, International PCTapplication No. WO 01/32711).

A fusion protein can be produced by standard recombinant techniques. Forexample, DNA fragments coding for the different polypeptide sequencescan be ligated together in-frame in accordance with conventionaltechniques, e.g., by employing blunt-ended or stagger-ended termini forligation, restriction enzyme digestion to provide for appropriatetermini, filling-in of cohesive ends as appropriate, alkalinephosphatase treatment to avoid undesirable joining, and enzymaticligation. In another embodiment, the fusion gene can be synthesized byconventional techniques including automated DNA synthesizers.Alternatively, PCR amplification of gene fragments can be carried outusing anchor primers that give rise to complementary overhangs betweentwo consecutive gene fragments that can subsequently be annealed andreamplified to generate a chimeric gene sequence (see, e.g., Ausubel etal. (eds.) Current Protocols in Molecular Biology, John Wiley & Sons,1992). Moreover, many expression vectors are commercially available thatalready encode a fusion moiety (e.g., a GST polypeptide). A FIX-encodingnucleic acid can be cloned into such an expression vector such that thefusion moiety is linked in-frame to the protease protein.

4. Polypeptide Modification

Modified FIX polypeptides can be prepared as unmodified (or naked)polypeptide chains or as posttranslationally modified polypeptides. Forsome applications, it can be desirable to prepare modified FIX in a“naked” form without post-translational or other chemical modifications.Naked polypeptide chains can be prepared in suitable hosts that do notpost-translationally modify FIX. Such polypeptides also can be preparedin in vitro systems and using chemical polypeptide synthesis. For otherapplications, particular modifications can be desired. In particular,for the purposes herein, glycosylation of the modified FIX polypeptidesto produce hyperglycosylated FIX polypeptides is preferred. Suchglycosylation can be performed in vivo using an appropriate expressionsystem, such as a mammalian expression system, in vitro (see e.g. Mikamiet al. (2006) J. Biotechnol. 127:65-78), or a combination of in vivo andin vitro methods in which, for example, the FIX polypeptide is expressedin prokaryotic cells and further modified in vitro using enzymatictransglycosylation (see e.g. Schwarz et al., (2010) Nature Chem. Biol.6:264-266). Additionally, pegylation, albumination, carboxylation,hydroxylation, phosphorylation, or other known modifications can bedesired. Modifications can be made in vitro or, for example, byproducing the modified FIX in a suitable host that produces suchmodifications.

5. Nucleotide Sequences

Nucleic acid molecules encoding FIX or modified FIX polypeptides areprovided herein. Nucleic acid molecules include allelic variants orsplice variants of any encoded FIX polypeptide. Exemplary of nucleicacid molecules provided herein are any that encode a modified FIXpolypeptide provided herein, such as any encoding a polypeptide setforth in any of SEQ ID NOS:75-272. In one embodiment, nucleic acidmolecules provided herein have at least 50, 60, 65, 70, 75, 80, 85, 90,91, 92, 93, 94, 95, or 99% sequence identity or hybridize underconditions of medium or high stringency along at least 70% of thefull-length of any nucleic acid encoding a FIX polypeptide providedherein. For example, the nucleic acid molecules provided herein have atleast or at least about 50, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94,95, or 99% sequence identity to the nucleic acid sequence set forth inSEQ ID NO:1. In another embodiment, a nucleic acid molecule can includethose with degenerate codon sequences encoding any of the FIXpolypeptides provided herein.

F. ASSESSING MODIFIED FIX POLYPEPTIDE ACTIVITIES

The activities and properties of FIX polypeptides can be assessed invitro and/or in vivo. Assays for such assessment are known to those ofskill in the art and are known to correlate tested activities andresults to therapeutic and in vivo activities. In one example, FIXvariants can be assessed in comparison to unmodified and/or wild-typeFIX. Such assays can be performed in the presence or absence of FVIIIa,phospholipids and/or calcium. In vitro assays include any laboratoryassay known to one of skill in the art, such as for example, cell-basedassays including coagulation assays, binding assays, protein assays, andmolecular biology assays. In vivo assays include FIX assays in animalmodels as well as administration to humans. In some cases, activity ofFIX polypeptides in vivo can be determined by assessing blood, serum, orother bodily fluid for assay determinants. FIX variants, such as thoseprovided herein, also can be tested in vivo to assess an activity orproperty, such as therapeutic effect.

Typically, assays described herein are with respect to the two-chainactivated form of FIX, i.e. FIXa. FIX polypeptides that have beenactivated via proteolytic cleavage after R145 and R180 can be preparedin vitro. The FIX polypeptides can be first prepared by any of themethods of production described herein, including, but not limited to,production in mammalian cells followed by purification. Cleavage of theFIX polypeptides into the active protease form of FIX can beaccomplished by incubation with activated factor XI (FXIa). Theactivated polypeptides can be used in any of the assays to measure FIXactivities described herein. Such assays also can be performed with thesingle chain zymogen form. For example, a single chain zymogen FIXpolypeptide can provide a negative control since such a form typicallydoes not exhibit the proteolytic or catalytic activity required for thecoagulant activity of FIX. In addition, such assays also can beperformed in the presence of cofactors, such as FVIIIa, and othermolecules, such as phospholipids and/or calcium, which in can augmentthe activity of FIX.

1. In Vitro Assays

Exemplary in vitro assays include assays to assess polypeptidemodification and activity. Modifications can be assessed using in vitroassays that assess glycosylation, γ-carboxylation and otherpost-translational modifications, protein assays and conformationalassays known in the art. Assays for activity include, but are notlimited to, measurement of FIX interaction with other coagulationfactors, such as FVIIIa and factor X, proteolytic assays to determinethe proteolytic activity of FIX polypeptides, assays to determine thebinding and/or affinity of FIX polypeptides for phosphatidylserines andother phospholipids, and cell based assays to determine the effect ofFIX polypeptides on coagulation.

Concentrations of modified FIX polypeptides can be assessed by methodswell-known in the art, including but not limited to, enzyme-linkedimmunosorbant assays (ELISA), SDS-PAGE; Bradford, Lowry, BCA methods; UVabsorbance, and other quantifiable protein labeling methods, such as,but not limited to, immunological, radioactive and fluorescent methodsand related methods. Assessment of cleavage products of proteolysisreactions, including cleavage of FIX polypeptides or products producedby FIX protease activity, can be performed using methods including, butnot limited to, chromogenic substrate cleavage, HPLC, SDS-PAGE analysis,ELISA, Western blotting, immunohistochemistry, immunoprecipitation,NH₂-terminal sequencing, fluorescence, and protein labeling.

Structural properties of modified FIX polypeptides can also be assessed.For example, X-ray crystallography, nuclear magnetic resonance (NMR),and cryoelectron microscopy (cryo-EM) of modified FIX polypeptides canbe performed to assess three-dimensional structure of the FIXpolypeptides and/or other properties of FIX polypeptides, such as Ca²⁺or cofactor binding.

Additionally, the presence and extent of FIX degradation can be measuredby standard techniques such as sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE), and Western blotting of electrophoresedFIX-containing samples. FIX polypeptides that have been exposed toproteases also can be subjected to N-terminal sequencing to determinelocation or changes in cleavage sites of the modified FIX polypeptides.

a. Glycosylation

FIX polypeptides can be assessed for the presence of glycosylation usingmethods well known in the art. Glycosylation of a polypeptide can beencharacterized from its enzymatically or chemically released carbohydratepool, using a wide variety of methods, such as high pH anion exchangechromatography (Townsend et al., (1991) Glycobiology 1:139-147), orfluorophore-assisted carbohydrate electrophoresis (FACE) (Kumar et al.,(1996) Biotechnol. Appl. Biochem. 24:207-214.), sequentialexoglycosidase digestions (Watzlawick et al., (1992) Biochemistry31:12198-12203; Tyagarajan et al., (1996) Glycobiology, 6:83-93), massspectrometry (Gillece-Castro et al., (1990) Meth. Enzymol. 193: 689-712;Duffin et al., (1992) Anal. Chem. 64:1440-1448; Papac et al., (1997) inTechniques in Glycobiology (Townsend R. R. and Hotchkiss A. T. eds.)Marcel Decker, Inc., New York, pp. 33-52; Fu et al., (1994) Carbohydr.Res. 261:173-186) and NMR (Fu et al., (1994) Carbohydr. Res.261:173-186).

For example, chemical release can be effected by hydrazinolysis, whichreleases N- and O-linked glycans from glycoproteins by incubation withanhydrous hydrazine. Enzymatic release can be effected by theendoglycosidases peptide N-glycosidase F (PNGase F), which removesunaltered most of the common N-linked carbohydrates from the polypeptidewhile hydrolyzing the originally glycosylated Asn residue to Asp.Hydrazinolysis or endoglycosidase treatment of FIX polypeptidesgenerates a reducing terminus that can be tagged with a fluorophore orchromophore label. Labeled FIX polypeptides can be analyzed byfluorophore-assisted carbohydrate electrophoresis (FACE). Thefluorescent tag for glycans also can be used for monosaccharideanalysis, profiling or fingerprinting of complex glycosylation patternsby HPLC. Exemplary HPLC methods include hydrophilic interactionchromatography, electronic interaction, ion-exchange, hydrophobicinteraction, and size-exclusion chromatography. Exemplary glycan probesinclude, but are not limited to, 3-(acetylamino)-6-aminoacridine (AA-Ac)and 2-aminobenzoic acid (2-AA). Carbohydrate moieties can also bedetected through use of specific antibodies that recognize theglycosylated FIX polypeptide.

In one method, mass spectrometry is used to assess site-specificcarbohydrate heterogeneity. This can involve matrix-assisted laserdesorption ionization mass spectrometry of collected HPLC-fractions(Sutton et al., (1994) Anal. Biochem. 218:34-46; Ploug et al., (1998) J.Biol. Chem. 273:13933-13943), or reversed phase HPLC directly coupledwith electrospray ionization mass spectrometry (LC/ESIMS) (see, e.g.,Huddleston et al., (1993) Anal. Chem. 65:877-884; Medzihradsky et al.,(2008) Methods Mol. Biol. 446:293-316). In one example, glycosylation atpotential N-glycosylation sites, such as an asparagine residue within anAsn-X-Ser/Thr/Cys motif, is assessed by LC/ESIMS. The potentialN-glycosylation sites in a FIX polypeptide can be identified, and aproteolytic enzyme can be selected that would separate these sites onindividual peptides. The digestion mixture is then analyzed by LC/ESIMS,a method that generates diagnostic carbohydrate ions by collisionalactivation (33). These diagnostic carbohydrate ions include, forexample, characteristic nonreducing end oxonium ions at m/z 204, 274 and292, 366, and 657, which indicate the presence of N-acetylhexosamine,neuraminic (sialic) acid, hexosyl-N-acetylhexosamine, andsialyl-hexosyl-Nacetylhexosamine, respectively. In addition toidentifying the presence of these ions by selective ion monitoring(SIM), the LC/ESIMS method also analyzes the peptide to assess themolecular weight, which can be used to indicate which peptide, and,therefore, which potential N-glycosylation site, contains thecarbohydrate.

b. Other Post-Translational Modifications

FIX polypeptides can be assessed for the presence of post-translationalmodifications other than glycosylation. Such assays are known in the artand include assays to measure hydroxylation, sulfation, phosphorylationand carboxylation. An exemplary assay to measure β-hydroxylationcomprises reverse phase HPLC analysis of FIX polypeptides that have beensubjected to alkaline hydrolysis (Przysiecki et al. (1987) PNAS84:7856-7860). Carboxylation and γ-carboxylation of FIX polypeptides canbe assessed using mass spectrometry with matrix-assisted laserdesorption ionization time-of-flight (MALDI-TOF) analysis, as describedfor other vitamin K-dependent polyppetides (se, e.g. Harvey et al. JBiol Chem 278:8363-8369, Maun et al. Prot Sci 14:1171-1180). Theinteraction of a FIX polypeptide containing the propeptide (pro-FIX)with the carboxylase responsible for post-translational γ-carboxylatemodification also can be assessed. The dissociation constant (K_(d))following incubation of carboxylase with flourescein-labeled pro-FIXpolypeptides can be measured by determining the amount of boundcarboxylase by anisotropy (Lin et al. (2004) J Biol Chem 279:6560-6566).Other exemplary assays to measure carboxylation include reverse phaseHPLC analysis of FIX polypeptides that have been subjected to alkalinehydrolysis (Przysiecki et al. (1987) PNAS 84: 7856-7860).

Exemplary assays to measure phosphorylation include use ofphosphospecific antibodies to phospho-serine and/or -tyrosine amino acidresidues or to a serine-phosphorylated FIX polypeptide. ³²P metaboliclabeling of cells that produce the FIX polypeptide also can be used toassess phosphorylation, wherein the labeled FIX polypeptide can bepurified and analyzed for incorporation of radioactive phosphate. Anexemplary assay for tyrosine sulfation includes ³⁵S labeling of cellsthat produce the FIX polypeptide. In such method, cells are incubatedwith either ³⁵S—S₂SO₄ or ³⁵S-methionine and incorporation of the ³⁵S isdetermined by normalization to the ³⁵S-methionine sample.

c. Proteolytic Activity

Modified FIX polypeptides can be tested for proteolytic activity towardsboth synthetic substrates and it's natural substrate, Factor X.Activated forms of the modified FIX polypeptides (FIXa) typically areused in the assay. Assays using a synthetic substrate, such as aCH₃SO₂-LGR-pNA peptide, can be employed to measure enzymatic cleavageactivity of the FIXa polypeptides. Hydrolysis of CH₃SO₂-LGR-pNA in thepresence of FIXa can be measured by assessing the production ofp-nitroanaline (pNA) from the cleavage reaction sample. The amount ofpNA in the sample is proportional to the absorbance of the sample at 405nm and thus indicates the extent of proteolytic activity in the FIXasample. Additional exemplary fluorogenic substrates that can be used toassess FIXa cleavage activity include, but are not limited to,Mes-D-CHD-Gly-Arg-AMC (Pefafluor FIXa10148) and H-D-Leu-PHG-Arg-AMC(Pefafluor FIXa3688), wherein cleavage is assessed by release of AMC,and the fluorogenic ester substrate, 4-methylumbelliferylp′-guanidinobenzoate (MUGB), where cleavage is assessed by the releaseof 4-methylumbelliferone fluorophore (4-MU) (see e.g. Example 3).Molecules that enhance FIXa catalytic activity, such as ethylene glycol,can be employed in such assays (Sturzebecher et al. (1997) FEBS Lett.(412) 295-300).

Proteolytic activity of FIXa also can be assessed by measuring theconversion of factor X (FX) into activated factor X (FXa), such asdescribed in Example 4, below. FIXa polypeptides, including the modifiedFIX polypeptides provided herein, can be incubated with FX polypeptidesin the presence of FVIIIa, phospholipids vesicles (phosphatidylserineand/or phosphatidylcholine) and Ca²⁺, and cleavage of FX to produce FXacan be assayed using a fluorogenic substrate, such as Spectrafluor FXa(CH₃SO₂-D-CHA-Gly-Arg-AMC), or a chromogenic substrate, such as S2222 orS2765 (Chromogenics AB, Molndal, Sweden), which are specifically cleavedby FXa.

d. Coagulation Activity

FIX polypeptides can be tested for coagulation activity by using assayswell known in the art. For example, some of the assays include, but arenot limited to, a two stage clotting assay (Liebman et al., (1985) PNAS82:3879-3883); the prothrombin time assay (PT, which can measureTF-dependent activity of FIXa in the extrinsic pathway); assays whichare modifications of the PT test; the activated partial thromboplastintime (aPTT, which can measure TF-independent activity of FIXa);activated clotting time (ACT); recalcified activated clotting time; theLee-White Clotting time; or thromboelastography (TEG) (Pusateri et al.(2005) Critical Care 9:S15-S24). For example, coagulation activity of amodified FIX polypeptide can be determined by a PT-based assay where FIXis diluted in FIX-deficient plasma, and mixed with prothrombin timereagent (recombinant TF with phospholipids and calcium), such as thatavailable as Innovin™ from Dade Behring. Clot formation is detectedoptically and time to clot is determined and compared againstFIX-deficient plasma alone. In vivo coagulation assays in animal models,such as those described below, also can be performed to assess thecoagulation activity of FIX polypeptides.

e. Binding to and/or Inhibition by Other Proteins and Molecules

Inhibition assays can be used to measure resistance of modified FIXpolypeptides to FIX inhibitors, such as, for example, antithrombin III(AT-III), heparain, AT-III/heparin complex, p-aminobenzamidine, serineprotease inhibitors, and FIX-specific antibodies. Assessment ofinhibition to other inhibitors also can be tested and include, but arenot limited to, other serine protease inhibitors Inhibition can beassessed by incubation of the inhibitor with FIX polypeptides that havebeen preincubated with and/or without FVIIIa. The activity of FIX canthen be measured using any one or more of the activity or coagulationassays described above, and inhibition by the inhibitor can be assessedby comparing the activity of FIX polyeptides incubated with theinhibitor, with the activity of FIX polypeptides that were not incubatedwith the inhibitor. For example, the inhibition of modified FIXpolypeptides by AT-III/heparin can be assessed as described in Example5, below Inhibition of wild-type FIXa or FIXa variants by theAT-III/heparin complex is assessed by incubating AT-III/heparin withFIXa and the measuring the catalytic activity of FIXa towards a smallmolecule substrate, Mesyl-D-CHG-Gly-Arg-AMC (Pefafluor FIXa;Pentapharm). Such assays can be performed in the presence or absence ofFVIIIa.

FIX polypeptides also can be tested for binding to other coagulationfactors and inhibitors. For example, FIX direct and indirectinteractions with cofactors, such as FVIIIa, substrates, such as FX andFIX, and inhibitors, such as antithrombin III and heparin, can beassessed using any binding assay known in the art, including, but notlimited to, immunoprecipitation, column purification, non-reducingSDS-PAGE, BIAcore® assays, surface plasmon resonance (SPR), fluorescenceresonance energy transfer (FRET), fluorescence polarization (FP),isothermal titration calorimetry (ITC), circular dichroism (CD), proteinfragment complementation assays (PCA), Nuclear Magnetic Resonance (NMR)spectroscopy, light scattering, sedimentation equilibrium, small-zonegel filtration chromatography, gel retardation, Far-western blotting,fluorescence polarization, hydroxyl-radical protein footprinting, phagedisplay, and various two-hybrid systems.

e. Phospholipid Affinity

Modified FIX polypeptide binding and/or affinity for phosphatidlyserine(PS) and other phospholipids can be determined using assays well knownin the art. Highly pure phospholipids (for example, known concentrationsof bovine PS and egg phosphatidylcholine (PC), which are commerciallyavailable, such as from Sigma, in organic solvent can be used to preparesmall unilamellar phospholipid vesicles. FIX polypeptide binding tothese PS/PC vesicles can be determined by relative light scattering at90° to the incident light. The intensity of the light scatter with PC/PSalone and with PC/PS/FIX is measured to determine the dissociationconstant (Harvey et al., (2003) J. Biol. Chem. 278:8363-8369). Surfaceplasma resonance, such as on a BIAcore biosensor instrument, also can beused to measure the affinity of FIX polypeptides for phospholipidmembranes (Sun et al., (2003) Blood 101:2277-2284).

2. Non-Human Animal Models

Non-human animal models can be used to assess activity and stability ofmodified FIX polypeptides. For example, non-human animals can be used asmodels for a disease or condition. Non-human animals can be injectedwith disease and/or phenotype-inducing substances prior toadministration of FIX variants to monitor the effects on diseaseprogression. Genetic models also are useful. Animals, such as mice, canbe generated which mimic a disease or condition by the overexpression,underexpression or knock-out of one or more genes. Such animals can begenerated by transgenic animal production techniques well-known in theart or using naturally-occurring or induced mutant strains. Examples ofuseful non-human animal models of diseases associated with FIX include,but are not limited to, models of bleeding disorders, in particularhemophilia. These non-human animal models can be used to monitoractivity of FIX variants compared to a wild type FIX polypeptide.

Animal models also can be used to monitor stability, half-life,clearance, and other pharmacokinetic and pharmacodynamic properties ofmodified FIX polypeptides. Such assays are useful for comparing modifiedFIX polypeptides and for calculating doses and dose regimens for furthernon-human animal and human trials. For example, a modified FIXpolypeptide can be injected into the tail vein of mice. Blood samplesare then taken at time-points after injection (such as minutes, hoursand days afterwards) and then the pharmacokinetic and pharmacodynamicproperties of the modified FIX polypeptides assessed, such as bymonitoring the serum or plasma at specific time-points for FIXa activityand protein concentration by ELISA or radioimmunoassay (see e.g. Example6). Blood samples also can be tested for coagulation activity inmethods, such as the aPTT assay (see e.g. Example 6).

Modified FIX polypeptides can be tested for therapeutic effectivenessusing animal models for hemophilia. In one non-limiting example, ananimal model such as a mouse can be used. Mouse models of hemophilia areavailable in the art and include FIX deficient mice (such as thoseutilized in Example 7, below) and mice expressing mutant FIXpolypeptides, and can be employed to test modified FIX polypeptides(Wang et al., (1997) PNAS 94:11563-11566; Lin et al., (1997) Blood90:3962-3966; Kundu et al., (1998) Blood 92: 168-174; Sabatino et al.,(2004) Blood 104(9):2767-2774; and Jin et al., (2004) Blood104:1733-1739; see also Example 7).

Other models of FIX deficiencies include hemophilic dogs that expressdefective FIX or that have been hepatectomized (Evans et al., (1989)PNAS 86:10095; Mauser et al., (1996) Blood 88:3451; and Kay et al.,(1994) PNAS 91:2353-2357).

3. Clinical Assays

Many assays are available to assess activity of FIX for clinical use.Such assays can include assessment of coagulation, protein stability,and half-life in vivo and phenotypic assays. Phenotypic assays andassays to assess the therapeutic effect of FIX treatment includeassessment of blood levels of FIX (such as measurement of serum FIXprior to administration and time-points following administrationsincluding, after the first administration, immediately after lastadministration, and time-points in between, correcting for the body massindex (BMI)), phenotypic response to FIX treatment includingamelioration of symptoms over time compared to subjects treated with anunmodified and/or wild type FIX or placebo. Examples of clinical assaysto assess FIX activity can be found such as in Franchini et al., (2005)Thromb Haemost. 93(6):1027-1035; Shapiro et al., (2005) Blood105(2):518-525; and White et al., (1997) Thromb. Haemost. 78(1):261-265.Patients can be monitored regularly over a period of time for routine orrepeated administrations, following administration in response to acuteevents, such as hemorrhage, trauma, or surgical procedures.

G. Formulation and Administration

Compositions for use in treatment of bleeding disorders are providedherein. Such compositions contain a therapeutically effective amount ofa Factor IX polypeptide as described herein. Effective concentrations ofFIX polypeptides or pharmaceutically acceptable derivatives thereof aremixed with a suitable pharmaceutical carrier or vehicle for systemic,topical or local administration. Compounds are included in an amounteffective for treating the selected disorder. The concentration ofactive compound in the composition will depend on absorption,inactivation, excretion rates of the active compound, the dosageschedule, and amount administered as well as other factors known tothose of skill in the art.

Pharmaceutical carriers or vehicles suitable for administration of thecompounds provided herein include any such carriers known to thoseskilled in the art to be suitable for the particular mode ofadministration. Pharmaceutical compositions that include atherapeutically effective amount of a FIX polypeptide described hereinalso can be provided as a lyophilized powder that is reconstituted, suchas with sterile water, immediately prior to administration.

1. Formulations

Pharmaceutical compositions containing a modified FIX can be formulatedin any conventional manner by mixing a selected amount of thepolypeptide with one or more physiologically acceptable carriers orexcipients. Selection of the carrier or excipient is within the skill ofthe administering profession and can depend upon a number of parameters.These include, for example, the mode of administration (i.e., systemic,oral, nasal, pulmonary, local, topical, or any other mode) and disordertreated. The pharmaceutical compositions provided herein can beformulated for single dosage (direct) administration or for dilution orother modification. The concentrations of the compounds in theformulations are effective for delivery of an amount, uponadministration, that is effective for the intended treatment. Typically,the compositions are formulated for single dosage administration. Toformulate a composition, the weight fraction of a compound or mixturethereof is dissolved, suspended, dispersed, or otherwise mixed in aselected vehicle at an effective concentration such that the treatedcondition is relieved or ameliorated.

The modified FIX polypeptides provided herein can be formulated foradministration to a subject as a two-chain FIXa protein. The modifiedFIX polypeptides can be activated by any method known in the art priorto formulation. For example, FIX can be activated by incubation withFXIa, such as FXIa immobilized on beads. Calcium can be included inthese processes to ensure full activation and correct folding of themodified FIXa protein. The modified FIX polypeptides provided hereinalso can be formulated for administration as a single chain protein. Themodified FIX polypeptides provided herein can be formulated such thatthe single-chain and two-chain forms are contained in the pharmaceuticalcomposition, in any ratio by appropriate selection of the medium toeliminate or control autoactivation.

The compound can be suspended in micronized or other suitable form orcan be derivatized to produce a more soluble active product. The form ofthe resulting mixture depends upon a number of factors, including theintended mode of administration and the solubility of the compound inthe selected carrier or vehicle. The resulting mixtures are solutions,suspensions, emulsions and other such mixtures, and can be formulated asan non-aqueous or aqueous mixture, creams, gels, ointments, emulsions,solutions, elixirs, lotions, suspensions, tinctures, pastes, foams,aerosols, irrigations, sprays, suppositories, bandages, or any otherformulation suitable for systemic, topical or local administration. Forlocal internal administration, such as, intramuscular, parenteral orintra-articular administration, the polypeptides can be formulated as asolution suspension in an aqueous-based medium, such as isotonicallybuffered saline or are combined with a biocompatible support orbioadhesive intended for internal administration. The effectiveconcentration is sufficient for ameliorating the targeted condition andcan be empirically determined. To formulate a composition, the weightfraction of compound is dissolved, suspended, dispersed, or otherwisemixed in a selected vehicle at an effective concentration such that thetargeted condition is relieved or ameliorated.

Generally, pharmaceutically acceptable compositions are prepared in viewof approvals for a regulatory agency or other prepared in accordancewith generally recognized pharmacopeia for use in animals and in humans.Pharmaceutical compositions can include carriers such as a diluent,adjuvant, excipient, or vehicle with which an isoform is administered.Such pharmaceutical carriers can be sterile liquids, such as water andoils, including those of petroleum, animal, vegetable or syntheticorigin, such as peanut oil, soybean oil, mineral oil, and sesame oil.Water is a typical carrier when the pharmaceutical composition isadministered intravenously. Saline solutions and aqueous dextrose andglycerol solutions also can be employed as liquid carriers, particularlyfor injectable solutions. Compositions can contain along with an activeingredient: a diluent such as lactose, sucrose, dicalcium phosphate, orcarboxymethylcellulose; a lubricant, such as magnesium stearate, calciumstearate and talc; and a binder such as starch, natural gums, such asgum acacia, gelatin, glucose, molasses, polvinylpyrrolidine, cellulosesand derivatives thereof, povidone, crospovidones and other such bindersknown to those of skill in the art. Suitable pharmaceutical excipientsinclude starch, glucose, lactose, sucrose, gelatin, malt, rice, flour,chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodiumchloride, dried skim milk, glycerol, propylene, glycol, water, andethanol A composition, if desired, also can contain minor amounts ofwetting or emulsifying agents, or pH buffering agents, for example,acetate, sodium citrate, cyclodextrine derivatives, sorbitanmonolaurate, triethanolamine sodium acetate, triethanolamine oleate, andother such agents. These compositions can take the form of solutions,suspensions, emulsion, tablets, pills, capsules, powders, and sustainedrelease formulations. Capsules and cartridges of e.g., gelatin for usein an inhaler or insufflator can be formulated containing a powder mixof a therapeutic compound and a suitable powder base such as lactose orstarch. A composition can be formulated as a suppository, withtraditional binders and carriers such as triglycerides. Oral formulationcan include standard carriers such as pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate, and other such agents. Preparations for oraladministration also can be suitably formulated with protease inhibitors,such as a Bowman-Birk inhibitor, a conjugated Bowman-Birk inhibitor,aprotinin and camostat. Examples of suitable pharmaceutical carriers aredescribed in “Remington's Pharmaceutical Sciences” by E. W. Martin. Suchcompositions will contain a therapeutically effective amount of thecompound, generally in purified form, together with a suitable amount ofcarrier so as to provide the form for proper administration to a subjector patient.

The formulation should suit the mode of administration. For example, themodified FIX can be formulated for parenteral administration byinjection (e.g., by bolus injection or continuous infusion). Theinjectable compositions can take such forms as suspensions, solutions oremulsions in oily or aqueous vehicles. The sterile injectablepreparation also can be a sterile injectable solution or suspension in anon-toxic parenterally-acceptable diluent or solvent, for example, as asolution in 1,4-butanediol. Sterile, fixed oils are conventionallyemployed as a solvent or suspending medium. For this purpose any blandfixed oil can be employed, including, but not limited to, syntheticmono- or diglycerides, fatty acids (including oleic acid), naturallyoccurring vegetable oils like sesame oil, coconut oil, peanut oil,cottonseed oil, and other oils, or synthetic fatty vehicles like ethyloleate. Buffers, preservatives, antioxidants, and the suitableingredients, can be incorporated as required, or, alternatively, cancomprise the formulation.

The polypeptides can be formulated as the sole pharmaceutically activeingredient in the composition or can be combined with other activeingredients. The polypeptides can be targeted for delivery, such as byconjugation to a targeting agent, such as an antibody. Liposomalsuspensions, including tissue-targeted liposomes, also can be suitableas pharmaceutically acceptable carriers. These can be prepared accordingto methods known to those skilled in the art. For example, liposomeformulations can be prepared as described in U.S. Pat. No. 4,522,811.Liposomal delivery also can include slow release formulations, includingpharmaceutical matrices such as collagen gels and liposomes modifiedwith fibronectin (see, for example, Weiner et al., (1985) J. Pharm. Sci.74(9):922-5). The compositions provided herein further can contain oneor more adjuvants that facilitate delivery, such as, but are not limitedto, inert carriers, or colloidal dispersion systems. Representative andnon-limiting examples of such inert carriers can be selected from water,isopropyl alcohol, gaseous fluorocarbons, ethyl alcohol, polyvinylpyrrolidone, propylene glycol, a gel-producing material, stearylalcohol, stearic acid, spermaceti, sorbitan monooleate, methylcellulose,as well as suitable combinations of two or more thereof.

The active compound is included in the pharmaceutically acceptablecarrier in an amount sufficient to exert a therapeutically useful effectin the absence of undesirable side effects on the subject treated. Thetherapeutically effective concentration can be determined empirically bytesting the compounds in known in vitro and in vivo systems, such as theassays provided herein.

a. Dosages

The precise amount or dose of the therapeutic agent administered dependson the particular FIX polypeptide, the route of administration, andother considerations, such as the severity of the disease and the weightand general state of the subject. Local administration of thetherapeutic agent will typically require a smaller dosage than any modeof systemic administration, although the local concentration of thetherapeutic agent can, in some cases, be higher following localadministration than can be achieved with safety upon systemicadministration. If necessary, a particular dosage and duration andtreatment protocol can be empirically determined or extrapolated. Forexample, exemplary doses of recombinant and native FIX polypeptides canbe used as a starting point to determine appropriate dosages. Forexample, a recombinant FIX (rFIXa) polypeptide that has been activatedto rFIXa, BeneFIX® Factor IX has been administered to patients withhemophilia B for the treatment of hemorrhage as well as in prophylacticand surgical settings at various doses. Dosage and duration of treatmentwith recombinant FIX depends on the severity of the factor IXdeficiency, the location and extent of bleeding, and the patient'sclinical condition, age and recovery of factor IX. For example, patientswith severe Hemophilia B (FIX activity of <1 IU/dL; 1% of normalactivity (where 1 IU represents the activity of Factor IX in 1 mL ofnormal, pooled plasma) will require more transfused FIX than patientswith moderate (FIX activity of 1-5 IU/dL; 1-5% of normal activity), ormild (FIX activity of >5-<40 IU/mL; >5-<40% of normal activity)hemophilia B. The initial estimated dose of BeneFIX® Factor IX can bedetermined using the following formula: Required units=body weight(kg)×desired factor IX increase (IU/dL or % of normal)×reciprocal ofobserved recovery (IU/kg per IU/dL). In clinical studies with adult andpediatric (<15 years) patients, one IU of BeneFIX per kilogram of bodyweight increased the circulating activity of factor IX as follows:Adults: 0.8±0.2 IU/dL [range 0.4 to 1.2 IU/dL]; Pediatric: 0.7±0.3 IU/dL[range 0.2 to 2.1 IU/dL]. Thus, for adult patients:

the number of Factor IX IU required (IU)=body weight (kg)×desired factorIX increase (% or IU/dL)×1.3 (IU/kg per IU/dL),

and, for pediatric patients:

the number of Factor IX IU required (IU)=body weight (kg)×desired factorIX increase (% or IU/dL)×1.4 (IU/kg per IU/dL).

Table 11 sets forth the typical dosing used for various bleedingepisodes.

TABLE 11 Circulating FIX Dosing Duration of activity required IntervalTherapy Type of Hemorrhage (% or IU/dL) (hours) (days) Minor: 20-3012-24 1-2 Uncomplicated hemarthroses, superficial muscle, or soft tissueModerate: 25-50 12-24 Treat until Intramuscle or soft tissue bleedingstops with dissection, and healing mucous membranes, begins, aboutdental extractions, 2 to 7 days or hematuria Major:  50-100 12-24 7-10Pharynx, retropharynx, retroperitoneum, CNS, surgery

The modified FIX polypeptides provided herein can be effective atreduced dosage amounts and/or reduced frequencies compared to nativerecombinant FIX. For example, the modified FIX polypeptides providedherein can be administered at less frequent dosing intervals, such as 24hours, 36 hours, 48 hours, 60 hours or more. In other examples, fewerdoses of the modified FIX polypeptides can be administered. For example,the modified FIX polyppetides provided herein can be administered justonce to achieve coagulation. In some embodiments, the dosages ofmodified FIX are reduced compared to native FIX. For example, thedosages can be less than or about 1 IU/kg, 2 IU/kg, 3 IU/kg, 4 IU/kg, 5IU/kg, 6 IU/kg, 7 IU/kg, 8 IU/kg, 9 IU/kg, 10 IU/kg, 20 IU/kg, 30 IU/kg,40 IU/kg or 50 IU/kg, 60 IU/kg, 70 IU/kg, 80 IU/kg, 90 IU/kg, or 100IU/kg. The dose, duration of treatment and the interval betweeninjections will vary with the severity of the bleed and the response ofthe patient to the treatment, and can be adjusted accordingly. Factorssuch as the level of activity and half-life of the modified FIX incomparison to the unmodified FIX can be taken into account when makingdosage determinations. Particular dosages and regimens can beempirically determined. For example, a modified FIX polypeptide thatexhibits a longer half-life than an unmodified FIX polypeptide can beadministered at lower doses and/or less frequently than the unmodifiedFIX polypeptide. Similarly, the dosages required for therapeutic effectusing a modified FIX polypeptide that displays increased coagulantactivity compared with an unmodified FIX polypeptide can be reduced infrequency and amount. Particular dosages and regimens can be empiricallydetermined by one of skill in the art.

b. Dosage Forms

Pharmaceutical therapeutically active compounds and derivatives thereofare typically formulated and administered in unit dosage forms ormultiple dosage forms. Formulations can be provided for administrationto humans and animals in dosage forms that include, but are not limitedto, tablets, capsules, pills, powders, granules, sterile parenteralsolutions or suspensions, oral solutions or suspensions, and oil wateremulsions containing suitable quantities of the compounds orpharmaceutically acceptable derivatives thereof. Each unit dose containsa predetermined quantity of therapeutically active compound sufficientto produce the desired therapeutic effect, in association with therequired pharmaceutical carrier, vehicle or diluent. Examples of unitdose forms include ampoules and syringes and individually packagedtablets or capsules. In some examples, the unit dose is provided as alyophilized powder that is reconstituted prior to administration. Forexample, a FIX polypeptide can be provided as lyophilized powder that isreconstituted with a suitable solution to generate a single dosesolution for injection. In some embodiments, the lyophilized powder cancontain the FIX polypeptide and additional components, such as salts,such that reconstitution with sterile distilled water results in a FIXpolypeptide in a buffered or saline solution. Unit dose forms can beadministered in fractions or multiples thereof. A multiple dose form isa plurality of identical unit dosage forms packaged in a singlecontainer to be administered in segregated unit dose form. Examples ofmultiple dose forms include vials, bottles of tablets or capsules orbottles of pints or gallons. Hence, multiple dose form is a multiple ofunit doses that are not segregated in packaging.

2. Administration of Modified FIX Polypeptides

The FIX polypeptides provided herein (i.e. active compounds) can beadministered in vitro, ex vivo, or in vivo by contacting a mixture, suchas a body fluid or other tissue sample, with a FIX polypeptide. Forexample, when administering a compound ex vivo, a body fluid or tissuesample from a subject can be contacted with the FIX polypeptides thatare coated on a tube or filter, such as for example, a tube or filter ina bypass machine. When administering in vivo, the active compounds canbe administered by any appropriate route, for example, orally, nasally,pulmonary, parenterally, intravenously, intradermally, subcutaneously,intraarticularly, intracisternally, intraocularly, intraventricularly,intrathecally, intramuscularly, intraperitoneally, intratracheally ortopically, as well as by any combination of any two or more thereof, inliquid, semi-liquid or solid form and are formulated in a mannersuitable for each route of administration. The modified FIX polypeptidescan be administered once or more than once, such as twice, three times,four times, or any number of times that are required to achieve atherapeutic effect. Multiple administrations can be effected via anyroute or combination of routes, and can be administered hourly, every 2hours, every three hours, every four hours or more.

The most suitable route for administration will vary depending upon thedisease state to be treated, for example the location of the bleedingdisorder. Generally, the FIX polypeptides will be administered byintravenous bolus injection, with an administration (infusing) time ofapproximately 2-5 minutes. In other examples, desirable blood levels ofFIX can be maintained by a continuous infusion of the active agent asascertained by plasma levels. It should be noted that the attendingphysician would know how to and when to terminate, interrupt or adjusttherapy to lower dosage due to toxicity, or bone marrow, liver or kidneydysfunctions. Conversely, the attending physician would also know how toand when to adjust treatment to higher levels if the clinical responseis not adequate (precluding toxic side effects). In other examples, thelocation of the bleeding disorder might indicate that the FIXformulation is administered via alternative routes. For example, localadministration, including administration into the brain (e.g.,intraventricularly) might be performed when the patient is experiencingbleeding in this region. Similarly, for treatment of bleeding in thejoints, local administration by injection of the therapeutic agent intothe joint (i.e., intraarticularly, intravenous or subcutaneous means)can be employed. In other examples, topical administration of thetherapeutic agent to the skin, for example formulated as a cream, gel,or ointment, or administration to the lungs by inhalation orintratracheally, might be appropriate when the bleeding is localized tothese areas.

The instances where the modified FIX polypeptides are be formulated as adepot preparation, the long-acting formulations can be administered byimplantation (for example, subcutaneously or intramuscularly) or byintramuscular injection. Thus, for example, the therapeutic compoundscan be formulated with suitable polymeric or hydrophobic materials (forexample as an emulsion in an acceptable oil) or ion exchange resins, oras sparingly soluble derivatives, for example, as a sparingly solublesalt.

The compositions, if desired, can be presented in a package, in a kit ordispenser device, that can contain one or more unit dosage formscontaining the active ingredient. The package, for example, containsmetal or plastic foil, such as a blister pack. The pack or dispenserdevice can be accompanied by instructions for administration. Thecompositions containing the active agents can be packaged as articles ofmanufacture containing packaging material, an agent provided herein, anda label that indicates the disorder for which the agent is provided.

3. Administration of Nucleic Acids Encoding Modified FIX Polypeptides(Gene Therapy)

Also provided are compositions of nucleic acid molecules encoding themodified FIX polypeptides and expression vectors encoding them that aresuitable for gene therapy. Rather than deliver the protein, nucleic acidcan be administered in vivo, such as systemically or by other route, orex vivo, such as by removal of cells, including lymphocytes,introduction of the nucleic therein, and reintroduction into the host ora compatible recipient.

Modified FIX polypeptides can be delivered to cells and tissues byexpression of nucleic acid molecules. Modified FIX polypeptides can beadministered as nucleic acid molecules encoding modified FIXpolypeptides, including ex vivo techniques and direct in vivoexpression. Nucleic acids can be delivered to cells and tissues by anymethod known to those of skill in the art. The isolated nucleic acidsequences can be incorporated into vectors for further manipulation. Asused herein, vector (or plasmid) refers to discrete elements that areused to introduce heterologous DNA into cells for either expression orreplication thereof. Selection and use of such vehicles are well withinthe skill of the artisan.

Methods for administering modified FIX polypeptides by expression ofencoding nucleic acid molecules include administration of recombinantvectors. The vector can be designed to remain episomal, such as byinclusion of an origin of replication or can be designed to integrateinto a chromosome in the cell. Modified FIX polypeptides also can beused in ex vivo gene expression therapy using non-viral vectors. Forexample, cells can be engineered to express a modified FIX polypeptide,such as by integrating a modified FIX polypeptide encoding-nucleic acidinto a genomic location, either operatively linked to regulatorysequences or such that it is placed operatively linked to regulatorysequences in a genomic location. Such cells then can be administeredlocally or systemically to a subject, such as a patient in need oftreatment.

Viral vectors, include, for example adenoviruses, adeno-associatedviruses (AAV), poxviruses, herpes viruses, retroviruses and othersdesigned for gene therapy can be employed. The vectors can remainepisomal or can integrate into chromosomes of the treated subject. Amodified FIX polypeptide can be expressed by a virus, which isadministered to a subject in need of treatment. Viral vectors suitablefor gene therapy include adenovirus, adeno-associated virus (AAV),retroviruses, lentiviruses, vaccinia viruses and others noted above. Forexample, adenovirus expression technology is well-known in the art andadenovirus production and administration methods also are well known.Adenovirus serotypes are available, for example, from the American TypeCulture Collection (ATCC, Rockville, Md.). Adenovirus can be used exvivo, for example, cells are isolated from a patient in need oftreatment, and transduced with a modified FIX polypeptide-expressingadenovirus vector. After a suitable culturing period, the transducedcells are administered to a subject, locally and/or systemically.Alternatively, modified FIX polypeptide-expressing adenovirus particlesare isolated and formulated in a pharmaceutically-acceptable carrier fordelivery of a therapeutically effective amount to prevent, treat orameliorate a disease or condition of a subject. Typically, adenovirusparticles are delivered at a dose ranging from 1 particle to 10¹⁴particles per kilogram subject weight, generally between 10⁶ or 10⁸particles to 10¹² particles per kilogram subject weight. In somesituations it is desirable to provide a nucleic acid source with anagent that targets cells, such as an antibody specific for a cellsurface membrane protein or a target cell, or a ligand for a receptor ona target cell. FIX also can be targeted for delivery into specific celltypes. For example, adenoviral vectors encoding FIX polypeptides can beused for stable expression in nondividing cells, such as liver cells(Margaritis et al. (2004) J. Clin Invest 113:1025-1031). In anotherexample, viral or nonviral vectors encoding FIX polypeptides can betransduced into isolated cells for subsequent delivery. Additional celltypes for expression and delivery of FIX might include, but are notlimited to, fibroblasts and endothelial cells.

The nucleic acid molecules can be introduced into artificial chromosomesand other non-viral vectors. Artificial chromosomes, such as ACES (see,Lindenbaum et al., (2004) Nucleic Acids Res. 32(21):e172) can beengineered to encode and express the isoform. Briefly, mammalianartificial chromosomes (MACs) provide a means to introduce largepayloads of genetic information into the cell in an autonomouslyreplicating, non-integrating format. Unique among MACs, the mammaliansatellite DNA-based Artificial Chromosome Expression (ACE) can bereproducibly generated de novo in cell lines of different species andreadily purified from the host cells' chromosomes. Purified mammalianACEs can then be re-introduced into a variety of recipient cell lineswhere they have been stably maintained for extended periods in theabsence of selective pressure using an ACE System. Using this approach,specific loading of one or two gene targets has been achieved in LMTK(−)and CHO cells.

Another method for introducing nucleic acids encoding the modified FIXpolypeptides is a two-step gene replacement technique in yeast, startingwith a complete adenovirus genome (Ad2; Ketner et al. (1994) PNAS 91:6186-6190) cloned in a Yeast Artificial Chromosome (YAC) and a plasmidcontaining adenovirus sequences to target a specific region in the YACclone, an expression cassette for the gene of interest and a positiveand negative selectable marker. YACs are of particular interest becausethey permit incorporation of larger genes. This approach can be used forconstruction of adenovirus-based vectors bearing nucleic acids encodingany of the described modified FIX polypeptides for gene transfer tomammalian cells or whole animals.

The nucleic acids can be encapsulated in a vehicle, such as a liposome,or introduced into a cells, such as a bacterial cell, particularly anattenuated bacterium or introduced into a viral vector. For example,when liposomes are employed, proteins that bind to a cell surfacemembrane protein associated with endocytosis can be used for targetingand/or to facilitate uptake, e.g. capsid proteins or fragments thereoftropic for a particular cell type, antibodies for proteins which undergointernalization in cycling, and proteins that target intracellularlocalization and enhance intracellular half-life.

For ex vivo and in vivo methods, nucleic acid molecules encoding themodified FIX polypeptide is introduced into cells that are from asuitable donor or the subject to be treated. Cells into which a nucleicacid can be introduced for purposes of therapy include, for example, anydesired, available cell type appropriate for the disease or condition tobe treated, including but not limited to epithelial cells, endothelialcells, keratinocytes, fibroblasts, muscle cells, hepatocytes; bloodcells such as T lymphocytes, B lymphocytes, monocytes, macrophages,neutrophils, eosinophils, megakaryocytes, granulocytes; various stem orprogenitor cells, in particular hematopoietic stem or progenitor cells,e.g., such as stem cells obtained from bone marrow, umbilical cordblood, peripheral blood, fetal liver, and other sources thereof.

For ex vivo treatment, cells from a donor compatible with the subject tobe treated or the subject to be treated cells are removed, the nucleicacid is introduced into these isolated cells and the modified cells areadministered to the subject. Treatment includes direct administration,such as, for example, encapsulated within porous membranes, which areimplanted into the patient (see, e.g., U.S. Pat. Nos. 4,892,538 and5,283,187 each of which is herein incorporated by reference in itsentirety). Techniques suitable for the transfer of nucleic acid intomammalian cells in vitro include the use of liposomes and cationiclipids (e.g., DOTMA, DOPE and DC-Chol) electroporation, microinjection,cell fusion, DEAE-dextran, and calcium phosphate precipitation methods.Methods of DNA delivery can be used to express modified FIX polypeptidesin vivo. Such methods include liposome delivery of nucleic acids andnaked DNA delivery, including local and systemic delivery such as usingelectroporation, ultrasound and calcium-phosphate delivery. Othertechniques include microinjection, cell fusion, chromosome-mediated genetransfer, microcell-mediated gene transfer and spheroplast fusion.

In vivo expression of a modified FIX polypeptide can be linked toexpression of additional molecules. For example, expression of amodified FIX polypeptide can be linked with expression of a cytotoxicproduct such as in an engineered virus or expressed in a cytotoxicvirus. Such viruses can be targeted to a particular cell type that is atarget for a therapeutic effect. The expressed modified FIX polypeptidecan be used to enhance the cytotoxicity of the virus.

In vivo expression of a modified FIX polypeptide can include operativelylinking a modified FIX polypeptide encoding nucleic acid molecule tospecific regulatory sequences such as a cell-specific or tissue-specificpromoter. Modified FIX polypeptides also can be expressed from vectorsthat specifically infect and/or replicate in target cell types and/ortissues. Inducible promoters can be use to selectively regulate modifiedFIX polypeptide expression. An exemplary regulatable expression systemis the doxycycline-inducible gene expression system, which has been usedto regulate recombinant FIX expression (Srour et al., (2003) Thromb.Haemost. 90(3):398-405).

Nucleic acid molecules, as naked nucleic acids or in vectors, artificialchromosomes, liposomes and other vehicles can be administered to thesubject by systemic administration, topical, local and other routes ofadministration. When systemic and in vivo, the nucleic acid molecule orvehicle containing the nucleic acid molecule can be targeted to a cell.

Administration also can be direct, such as by administration of a vectoror cells that typically targets a cell or tissue. For example, tumorcells and proliferating can be targeted cells for in vivo expression ofmodified FIX polypeptides. Cells used for in vivo expression of anmodified FIX polypeptide also include cells autologous to the patient.Such cells can be removed from a patient, nucleic acids for expressionof an modified FIX polypeptide introduced, and then administered to apatient such as by injection or engraftment.

H. THERAPEUTIC USES

The modified FIX polypeptides and nucleic acid molecules provided hereincan be used for treatment of any condition for which unmodified FIX isemployed. Thus, for example, the modified FIX polypeptides can be usedas procoagulants for the treatment of bleeding disorders, includingcongenital and acquired bleeding disorders, such as hemophilia.Typically, therefore, the modified FIX polypeptides provided herein areprocoagulants that are used in the treatment of bleeding disorders. Inother particular examples, however, the modified FIX polypeptides can beused as anticoagulants. For example, a hyperglycosylated modified FIXpolypeptide that also contains one or more modifications that result ina lack of catalytic activity for it's substrate, FX, can be used as ananticoagulant to treat thrombotic disorders.

The modified FIX polypeptides provided herein have therapeutic activityalone or in combination with other agents. The modified polypeptidesprovided herein are designed to retain therapeutic activity but exhibitmodified properties, such as improved pharmacokinetic andpharmacodynamic properties, increased resistance to inhibitors and/orimproved catalytic activity. Such modified properties and activities,for example, can improve the therapeutic effectiveness of thepolypeptides. The modified FIX polypeptides and encoding nucleic acidmolecules provided herein can be used for treatment of any condition forwhich unmodified FIX is employed. This section provides exemplary usesof and administration methods. These described therapies are exemplaryonly and do not limit the applications of modified FIX polypeptides.

The modified FIX polypeptides provided herein can be used in varioustherapeutic as well as diagnostic methods in which FIX is employed. Suchmethods include, but are not limited to, methods of treatment ofphysiological and medical conditions described and listed below.Modified FIX polypeptides provided herein can exhibit improvement of invivo activities and therapeutic effects compared to wild-type FIX,including lower dosage to achieve the same effect, a more sustainedtherapeutic effect and other improvements in administration andtreatment.

The modified FIX polypeptides described herein can exhibit improvedpharmacokinetic and pharmacodynamic properties, increased catalyticactivity, increased resistance to inhibitors and/or increased coagulantactivity compared to an unmodified FIX polypeptide. Such polypeptidescan be used as procoagulants for the treatment of, for example, bleedingdisorders, including congenital bleeding disorders and acquired bleedingdisorders. In some examples, the modified FIX polypeptides providedherein that have non-native glycosylation sites also containmodifications that result in a modified FIX polypeptide that inhibitscoagulation, such that the modified FIX polypeptide is an anti-coagulantand can be used to treat, for example, thrombotic diseases anddisorders. The modified FIX polypeptides provided herein can be used todeliver longer-lasting, more stable therapies. Examples of therapeuticimprovements using modified FIX polypeptides include, but are notlimited to, lower dosages, fewer and/or less frequent administrations,decreased side effects and increased therapeutic effects.

Typically, the modified FIX polypeptides provided herein areprocoagulants and can be used to treat bleeding disorders, includingcongenital bleeding disorders and acquired bleeding disorders. Inparticular examples, modified FIX polypeptides are intended for use intherapeutic methods in which other modified and unmodified FIXpolypeptides have been used for treatment. Exemplary diseases anddisorders that can be treated with the modified FIX polypeptides, aloneor in combination with other agents, including other procoagulants,include, but are not limited to, blood coagulation disorders,hematologic diseases, hemorrhagic disorders, hemophilias, in particularhemophilia B, and acquired blood disorders, including bleedingassociated with trauma and surgery. In some embodiments, the bleedingsto be treated by FIX polypeptides occur in organs such as the brain,inner ear region, eyes, liver, lung, tumor tissue, gastrointestinaltract. In other embodiments, the bleeding is diffuse, such as inhemorrhagic gastritis and profuse uterine bleeding.

Patients with bleeding disorders, such as hemophilia, are often at riskfor hemorrhage and excessive bleeding during surgery, including dentalextraction, or trauma. Such patients often have acute haemarthroses(bleedings in joints), chronic hemophilic arthropathy, haematomas, (suchas, muscular, retroperitoneal, sublingual and retropharyngeal),bleedings in other tissue, haematuria (bleeding from the renal tract),cerebral hemorrhage, and gastrointestinal bleedings (such as, UGIbleeds), that can be treated with modified FIX polypeptides. Thus, incsome examples, the modified FIX polypeptides are used to treat bleedingepisodes due to trauma or surgery, or lowered count or activity ofplatelets, in a subject. Exemplary methods for patients undergoingsurgery include treatments to prevent hemorrhage and treatments before,during, or after surgeries.

Although typically the modified FIX polypeptides provided herein exhibitimproved coagulant activity compared to a modified FIX polypeptide, insome examples, the modified FIX polypeptides provided herein can containone or more non-native glycosylation sites and also lack functionalpeptidase activity. Such modified FIX polypeptides can be used intherapeutic methods to inhibit blood coagulation (see e.g., U.S. Pat.No. 6,315,995). Modified FIX polypeptides that inhibit blood coagulationcan be used in anticoagulant methods of treatment for ischemic andthrombotic disorders. In surgical patients with an increased risk ofexcessive clotting, such as patients with deep vein thrombosis (DVT) orsuperficial vein thrombosis (SVT), the modified FIX polypeptidesprovided herein that are anticoagulants can be administered to preventexcessive clotting in surgeries. In some cases treatment is performedwith FIX alone. In some cases, FIX is administered in conjunction withadditional anticoagulation factors as required by the condition ordisease to be treated.

Treatment of diseases and conditions with modified FIX polypeptides canbe effected by any suitable route of administration using suitableformulations as described herein including, but not limited to,injection, pulmonary, oral and transdermal administration. If necessary,a particular dosage and duration and treatment protocol can beempirically determined or extrapolated. For example, exemplary doses ofrecombinant and native FIX polypeptides, such as recommended dosages ofBeneFIX® Coagulation Factor IX (Recombinant) as described above, can beused as a starting point to determine appropriate dosages. Modified FIXpolypeptides that are hyperglycosylated and have an increased half-lifein vivo, or that have increased resistance to inhibitors, or haveincreased catalytic activity, can be effective at reduced dosage amountsand/or frequencies. Dosages and dosage regimens for unmodified FIXpolypeptides can be used as guidance for determining dosages for themodified FIX polypeptides provided herein. Factors such as the half-lifeand level of activity of the modified FIX in comparison to theunmodified FIX can be used in making such determinations. Particulardosages and regimens can be empirically determined.

Dosage levels and regimens can be determined based upon known dosagesand regimens, and, if necessary can be extrapolated based upon thechanges in properties of the modified polypeptides and/or can bedetermined empirically based on a variety of factors. Such factorsinclude body weight of the individual, general health, age, the activityof the specific compound employed, sex, diet, time of administration,rate of excretion, drug combination, the severity and course of thedisease, and the patient's disposition to the disease and the judgmentof the treating physician. The active ingredient, the polypeptide,typically is combined with a pharmaceutically effective carrier. Theamount of active ingredient that can be combined with the carriermaterials to produce a single dosage form or multi-dosage form can varydepending upon the host treated and the particular mode ofadministration.

The effect of the FIX polypeptides on the clotting time of blood can bemonitored using any of the clotting tests known in the art including,but not limited to, whole blood partial thromboplastin time (PTT), theactivated partial thromboplastin time (aPTT), the activated clottingtime (ACT), the recalcified activated clotting time, or the Lee-WhiteClotting time.

Upon improvement of a patient's condition, a maintenance dose of acompound or compositions can be administered, if necessary; and thedosage, the dosage form, or frequency of administration, or acombination thereof can be modified. In some cases, a subject canrequire intermittent treatment on a long-term basis upon any recurrenceof disease symptoms or based upon scheduled dosages. In other cases,additional administrations can be required in response to acute eventssuch as hemorrhage, trauma, or surgical procedures.

Hemophilia

Hemophilia is a bleeding disorder that is caused by a deficiency in oneor more blood coagulation factors. It is characterized by a decreasedability to form blood clots at sites of tissue damage. CongenitalX-linked hemophilias include hemophilia A and hemophilia B, or Christmasdisease, which are caused by deficiencies in FVIII and FIX,respectively. Hemophilia A occurs at a rate of 1 out of 10,0000 males,while hemophilia B occurs in 1 out of 50,000 males.

Patients with hemophilia suffer from recurring joint and muscle bleeds,which can be spontaneous or in response to trauma. The bleeding cancause severe acute pain, restrict movement, and lead to secondarycomplications including synovial hypertrophy. Furthermore, the recurringbleeding in the joints can cause chronic synovitis, which can causejoint damage, destroying synovium, cartilage, and bone.

The modified FIX polypeptides provided herein and the nucleic acidsencoding the modified FIX polypeptides provided herein can be used intherapies for hemophilia, including treatment of bleeding conditionsassociated with hemophilia. The modified FIX polypeptides providedherein can be used, for example, to control or prevent spontaneousbleeding episodes or to control or prevent bleeding in response totrauma or surgical procedures.

The modified FIX polypeptides herein can exhibit improvedpharmacokinetic and pharmacodynamic properties, such as improved serumhalf-life, increased resistance to inhibitors, increased catalyticactivity, and/or increased coagulant activity. Thus, modified FIXpolypeptides can be used to deliver longer lasting or otherwise improvedtherapies for hemophilia. Examples of therapeutic improvements usingmodified FIX polypeptides include for example, but are not limited to,lower dosages, fewer and/or less frequent administrations, decreasedside effects, and increased therapeutic effects.

Modified FIX polypeptides can be tested for therapeutic effectiveness,for example, by using animal models. For example FIX-deficient mice, orany other known disease model for hemophilia, can be treated withmodified FIX polypeptides. Progression of disease symptoms andphenotypes is monitored to assess the effects of the modified FIXpolypeptides. Modified FIX polypeptides also can be administered toanimal models as well as subjects such as in clinical trials to assessin vivo effectiveness in comparison to placebo controls and/or controlsusing unmodified FIX.

a. Hemophilia B

Hemophilia B can be effectively managed with administration of FIXtherapeutics. Patients with severe Hemophilia B have an FIX activity of<1 IU/dL (1% of normal activity), patients with moderate Hemophilia Bhave a FIX activity of 1-5 IU/dL (1-5% of normal activity) and patientswith mild hemophilia B have a FIX activity of >5-<40 IU/mL (>5-<40% ofnormal activity). With proper prophylactic replacement therapy and/ortreatment of particular bleeding episodes with an appropriate amount ofFIX, patients often can achieve normal life span. Administration of FIXcan aid in controlling bleeding during surgery, trauma, during dentalextraction, or to alleviate bleeding associated with hemarthroses,hematuria, mucocutaneous bleeding, such as epistaxis or gastrointestinaltract bleeding, cystic lesions in subperiosteal bone or soft tissue, orhematomas, which cause neurological complications such as intracranialbleeding, spinal canal bleeding. Death in patients with hemophilia isoften the result of bleeding in the central nervous system. Otherserious complications in hemophilic patients include development ofinhibitors to coagulation factor therapeutics and disease.

The most frequent alterations in the FIX gene in hemophilia B patientsare point mutations, in particular missense mutations. Most of theidentified FIX mutations occur in amino acid residues in the codingregion of the FIX gene, often affecting evolutionarily conserved aminoacids. The severity of the hemophilia depends upon the nature of themutation. Mutations in the coding region can affect a number ofdifferent properties or activities of the FIX polypeptide includingalteration of protease activity, cofactor binding, signal peptide orpropeptide cleavage, post-translational modifications, and inhibition ofcleavage of FIX into its activated form. Other types of point mutationsinclude nonsense mutations that produce an unstable truncated FIXpolypeptide, and frameshift mutations (small deletions and insertions)that result in a terminally aberrant FIX molecule. In addition, FIXpoint mutations can be found in the promoter region, which can disruptthe recognition sequences for several specific gene regulatory proteins,resulting in reduced transcription of coagulation factor IX. DecreasedFIX as a result of transcriptional abnormalities is called Hemophilia BLeyden. An exemplary mutation in the promoter region includes disruptionof the HNF-4 binding site, which affect regulation of FIX transcriptionby the androgen receptor. The severity of this type of hemophilia isgoverned by the levels of androgen in the blood, which increase duringpuberty and partially alleviate the FIX transcriptional deficiency(Kurachi et al. (1995)). Other missense nucleotide changes affect theprocessing of factor IX primary RNA transcript. For example, somemutations occur at evolutionarily conserved donor-splice (GT), andacceptor-splice (AG) consensus sequences, which can create crypticsplice junctions and disrupt assembly of spliceosomes. Some severe casesof hemophilia (approximately 10%) present with large deletions in theFIX gene.

Treatment of FIX deficiency, and thus hemophilia B, most often involvesadministration of FIX, including recombinant forms of FIX, purifiedplasma FIX preparations or purified plasma concentrates. Thus,similarly, the modified FIX polypeptides herein, and nucleic acidsencoding modified FIX polypeptides, can be used for treatment ofhemophilia B. The modified FIX polypeptides herein can exhibit improvedpharmacokinetic and pharmacodynamic properties, such as improved serumhalf-life, increased resistance to inhibitors, increased catalyticactivity, and/or increased coagulant activity. Thus, modified FIXpolypeptides can be used to deliver improved therapies for hemophilia.Examples of therapeutic improvements using modified FIX polypeptidesinclude for example, but are not limited to, lower dosages, fewer and/orless frequent administrations, decreased side effects, and increasedtherapeutic effects.

b. Hemophilia A

Hemophilia A, which accounts for approximately 85% of all cases ofhemophilia, results from mutations(s) in the factor VIII gene on the Xchromosome, leading to a deficiency or dysfunction of the FVIII protein.Typically, treatment of hemophilia A with native FIX polypeptides,including recombinant FIX polypeptides such as BeneFIX® CoagulationFactor IX (Recombinant), or plasma-purified FIX polypeptides is notrecommended because the native FIX polypeptide requires FVIIIa forcatalytic activity to effect coagulation. Modified FIX polypeptides,however, such as those described herein, that contain one or moremodifications to increase the FIX intrinsic activity, can be used in thetreatment of hemophilia B. Such polypeptides have FVIII-independentactivity, and thus can function as a coagulant in hemophilia A patients.For example, the modified FIX polypeptides described above, such asthose that contain one or more modifications to introduce or eliminateone or more non-native glycosylation sites, and/or one or moremodifications to increase resistance to AT-III and/or heparin, and thatalso contain and one or more modifications to increase activity of themodified FIX polypeptide in the absence of FVIIIa, can be used to treatbleeding episodes in patients with Hemophilia A.

Modifications to increase intrinsic activity of a FIX polypeptide suchthat it can act in a FVIIIa-independent manner are described above andelsewhere (see e.g. Hopfner et al., (1997) EMBO J. 16:6626-6635; Kolkmanet al., (2000) Biochem. 39:7398-7405; Sichler et al., (2003) J. Biol.Chem 278:4121-4126; Begbie et al., (2005) Thromb Haemost. 94(6):1138-47,U.S. Pat. No. 6,531,298 and U.S. Patent Publication Nos. 20080167219 and20080214461), and include, but are not limited to, amino acidreplacements V86A, V86N, V86D, V86E, V86Q, V86G, V86H, V86I, V86L, V86M,V86F, V86S, V86T, V86W, V86Y, Y259F, A261K, K265T, E277V, E277A, E277N,E277D, E277Q, E277G, E277H, E2771, E277L, E277M, E277F, E277S, E277T,E277W, E277Y, R338A, R338V, R3381, R338F, R338W, R338S, R338T, Y345F,I383V and E388G. For example, a modified FIX polypeptide provided hereincan contain the amino acid substitutions Y259F/K265T, Y259F/K265T/Y345F,Y259F/A261K/K265T/Y345F, Y259F/K265T/Y345F/I383V/E388G orY259F/A261K/K265T/Y345F/1383V/E388G and can exhibit increased intrinsicactivity. Such modified FIX polypeptides can be used, therefore, in thetreatment of Hemophilia A.

J. COMBINATION THERAPIES

Any of the modified FIX polypeptides, and nucleic acid moleculesencoding modified FIX polypeptides described herein can be administeredin combination with, prior to, intermittently with, or subsequent to,other therapeutic agents or procedures including, but not limited to,other biologics, small molecule compounds and surgery. For any diseaseor condition, including all those exemplified above, for FIX isindicated or has been used and for which other agents and treatments areavailable, FIX can be used in combination therewith. Hence, the modifiedFIX polypeptides provided herein similarly can be used. Depending on thedisease or condition to be treated, exemplary combinations include, butare not limited to combination with other plasma purified or recombinantcoagulation factors, procoagulants, anticoagulants, anti-coagulationantibodies, glycosaminoglycans, heparins, heparinoids, heparinderivatives, heparin-like drugs, coumarins, such as warfarin andcoumarin derivatives. Additional procoagulants that can be used incombination therapies with modified FIX polypeptides provided hereinthat have procoagulant properties include, but are not limited to,vitamin K, vitamin K derivatives, other coagulation factors, and proteinC inhibitors. Additional anticoagulants that can be used in combinationtherapies with modified FIX polypeptides provided herein that haveanticoagulant properties include, but are not limited to, β2adrenoreceptor antagonists, neuropeptide V2 antagonists, prostacyclinanalogs, thromboxane synthase inhibitors, calcium agonists, elastaseinhibitors, non-steroidal anti-inflammatory molecules, thrombininhibitors, lipoxygenase inhibitors, FVIIa inhibitors, FXa inhibitors,phosphodiesterase III inhibitors, fibrinogen, vitamin K antagonists, andglucoprotein IIb/IIIa antagonists.

K. ARTICLES OF MANUFACTURE AND KITS

Pharmaceutical compounds of modified FIX polypeptides for nucleic acidsencoding modified FIX polypeptides, or a derivative or a biologicallyactive portion thereof can be packaged as articles of manufacturecontaining packaging material, a pharmaceutical composition which iseffective for treating a FIX-mediated disease or disorder, and a labelthat indicates that modified FIX polypeptide or nucleic acid molecule isto be used for treating a FIX-mediated disease or disorder.

The articles of manufacture provided herein contain packaging materials.Packaging materials for use in packaging pharmaceutical products arewell known to those of skill in the art. See, for example, U.S. Pat.Nos. 5,323,907, 5,033,252 and 5,052,558, each of which is incorporatedherein in its entirety. Examples of pharmaceutical packaging materialsinclude, but are not limited to, blister packs, bottles, tubes,inhalers, pumps, bags, vials, containers, syringes, bottles, and anypackaging material suitable for a selected formulation and intended modeof administration and treatment. A wide array of formulations of thecompounds and compositions provided herein are contemplated as are avariety of treatments for any FIX-mediated disease or disorder.

Modified FIX polypeptides and nucleic acid molecules also can beprovided as kits. Kits can include a pharmaceutical compositiondescribed herein and an item for administration. For example a modifiedFIX can be supplied with a device for administration, such as a syringe,an inhaler, a dosage cup, a dropper, or an applicator. The kit can,optionally, include instructions for application including dosages,dosing regimens and instructions for modes of administration. Kits alsocan include a pharmaceutical composition described herein and an itemfor diagnosis. For example, such kits can include an item for measuringthe concentration, amount or activity of FIX or a FIX regulated systemof a subject.

The following examples are included for illustrative purposes only andare not intended to limit the scope of the invention.

L. EXAMPLES Example 1 Cloning and Expression of Factor IX PolypeptidesA. Cloning of FIX Gene

The nucleic acid encoding the 461 amino acid human FIX precursorpolypeptide (P00740; set forth in SEQ ID NO:1) was cloned into themammalian expression vector, pFUSE-hIgG1-Fc2 (abbreviated here as pFUSE)(InvivoGen; SEQ ID NO:23), which contains a composite promoter,hEF1-HTLV, comprising the Elongation Factor-1α (EF-1α) core promoter andthe R segment and part of the U5 sequence (R-U5′) of the human T-CellLeukemia Virus (HTLV) Type 1 Long Terminal Repeat. The In-Fusion CFDry-Down PCR Cloning Kit (Clontech) was used according to the conditionsspecified by the supplier.

For the In-Fusion process, plasmid pFUSE without the humanimmunoglobulin 1 (hIgG1) Fc portion was linearized using polymerasechain reaction (PCR) with the pFUSE-Acc-F1 forward primer:GTGCTAGCTGGCCAGACATGATAAG (SEQ ID NO:24) and the pFUSE-Acc-R3 reverseprimer: CATGGTGGCCCTCCTTCGCCGGTGATC (SEQ ID NO:25), and was used asAcceptor DNA. The full-length coding sequence of FIX was amplified byPCR using human FIX cDNA (Origene) as template with theFIX-wtsp-Invivo-F1 forward primer: CGAAGGAGGGCCACCATGCAGCGCGTGAACATGATC(SEQ ID NO:26) and FIX-Invivo-R1 reverse primer:TGTCTGGCCAGCTAGCACTTAAGTGAGCTTTGTTTTTTCC (SEQ ID NO:27). For two FIXDonor amplification primer sequences set forth above, both FIX ‘ATG’start and complementary sequence of ‘TAA’ stop codons are underlined inthe forward and reverse primer sequences, respectively. The 18-nt longhomology regions, a non-annealing 5′ primer tail for In-Fusion, areshown in bold. Standard PCR reaction and thermocycling conditions wereused in conjunction with the Phusion High-Fidelity Master Mix Kit (NewEngland Biolabs), as recommended by the manufacturer. Both Acceptor andDonor PCR products were then digested with DpnI restriction enzyme toremove E. coli-derived dam methylated PCR template backgrounds. Theywere then mixed together, and the In-Fusion reaction was run usingconditions specified by the supplier. The reaction mix was transformedinto E. coli XL1Blue supercompetent cells (Stratagene). Colonies wereselected on 2×YT agar plates supplemented with 25 ppm Zeocin(InvivoGen). Plasmid DNA was isolated from selected clones, andsequenced to verify correct cloning.

B. Generation of FIX Variants

FIX variants were generated using the QuikChange Lightning Site-DirectedMutagenesis Kit (Stratagene) according to manufacturer's instructionswith specifically designed oligonucleotides that served as primers toincorporate designed mutations into the newly synthesized DNA.Complementary primers that include the desired mutations were extendedduring cycling using purified, double-stranded super-coiled pFUSEplasmid DNA that contained the cloned FIX cDNA sequence as a template.Extension of the primers resulted in incorporation of the mutations ofinterest into the newly synthesized strands, and resulted in a mutatedplasmid with staggered nicks. Following amplification, the mutagenesisproduct was digested with DpnI restriction enzyme to remove dammethylated parental strands of the E. coli-derived pFUSE DNA. The DNAwas then transformed into E. coli XL1Blue supercompetent cells(Stratagene) followed by selection on 2×YT agar plates supplemented with25 ppm Zeocin (InvivoGen). Plasmid DNA was isolated from selectedclones, and sequenced to verify for incorporation of mutation(s) at thedesired location(s) on the FIX gene.

The nucleotide sequence of one of the oligonucleotides from eachcomplementary primer pair used to generate the FIX variants is providedin Table 12. The nucleotide triplet sequences that encode a substitutedamino acid are shown in uppercase. For example, to generate a FIXvariant containing the substitutions A103N/N105S (A[103]N/N[105]S bychymotrypsin numbering; SEQ ID NO:77), the A103N/N105S-Forward primer,and a primer that is complementary to A103N/N105S-Forward, were used toreplace a 9-bp ‘GCTgatAAC’ wild-type sequence with a 9-bp ‘AATgatAGC’mutant sequence (changed nucleotide triplets are denoted by upper case).

Table 12 below sets forth the oligonucleotide primers used for FIXmutagenesis. The mutant triplets are shown in upper case, and primernames correspond to the mutation, by chymotrypsin numbering, produced asa result of the mutagenesis using the primer.

TABLE 12 SEQ ID Primer Name Primer Sequence (5′ to 3′) NO. F9-A[103]N/gtaaaaatagtAATgatAGCaaggtggtttg  28 N[105]S-For F9-D[104]N/gtaaaaatagtgctAATaacAGTgtggtttgctcctgtactg  29 K[106]S-For F9-K[106]N/gtgctgataacAATgtgAGTtgctcctgtactg  30 V[108]S-For F9-D[85]N-ForgaactgtgaattaAATgtaacatgtaac  31 F9-T[148]A-ForctcacccgtgctgagGCTgtttttcctgatgtg  32 F9-D39N/F41T-ForgaatggtaaagttAATgcaACCtgtggaggctctatc  33 F9-K63N-ForgaaactggtgttAACattacagttgtcgc  34 F9-I86S-For gcgaaatgtgAGTcgaattattcctc 35 F9-A95bS-For caactacaatgcaAGTattaataagtacaac  36 F9-K243N-ForaaggaaaaaacaAATctcacttaagtgctagctg  37 F9-E240N-ForctggattaagAATaaaacaaagctc  38 F9-E74N-ForcaggtgaacataatattAACgagacagaacatacag  39 F9-T76N/H78S-ForgaacataatattgaggagAACgaaAGTacagagcaaaag  40 F9-K82N/N84S-ForcagaacatacagagcaaAATcgaTCTgtgattcgaattattc  41 F9-L153N-ForgggagatcagctAATgttcttcagtac  42 F9-F145N/H147S-ForctggggaagagtcAACTCCaaagggagatcag  43 F9-K222N/K224S-ForgagtgtgcaatgAACggcTCAtatggaatatatac  44 F9-S151N/L153S-ForcttccacaaagggagaAATgctTCAgttcttca  45 F9-N95S-ForcctcaccacaactacAGTgcagctattaataagtacaacc  46 F9-Y117N-ForcttagtgctaaacagcAACgttacacctatttgc  47 F9-G149N-ForggaagagtcttccacaaaAACagatcagctttagttc  48 F9-R150N/A152S-ForgtcttccacaaagggAACtcaTCTttagttcttcagtac  49 F9-R150A-ForgtcttccacaaagggGCAtcagctttagttcttcag  50 F9-R150E-ForgtcttccacaaagggGAAtcagctttagttcttcag  51 F9-R150Y-ForgtcttccacaaagggTACtcagctttagttcttcag  52 F9-R143Q-ForgtaagtggctggggaCAAgtcttccacaaaggg  53 F9-R143A-ForgtaagtggctggggaGCAgtcttccacaaaggg  54 F9-R143Y-ForgtaagtggctggggaTACgtcttccacaaaggg  55 F9-R143L-ForgtaagtggctggggaCTGgtcttccacaaaggg  56 F9-V38M-ForgttttgaatggtaaaATGgatgcattctgtggaggc  57 F9-V38Y-ForgttttgaatggtaaaTACgatgcattctgtggaggc  58 F9-D39M-ForgttttgaatggtaaagttATGgcattctgtggaggc  59 F9-D39Y-ForgttttgaatggtaaagttTACgcattctgtggaggc  60 F9-A40M-ForgttttgaatggtaaagttgatATGttctgtggaggctctatc  61 F9-A40Y-ForgttttgaatggtaaagttgatTACttctgtggaggctctatc  62 F9-R233A/K230A-ForcaaatatggaatatataccGCAgtatccGCAtatgtcaactg  63 gattaagF9-R233E/K230E-For caaatatggaatatataccGAAgtatccGAAtatgtcaactg  64gattaag F9-R233A-For gaatatataccaaggtatccGCAtatgtcaactggattaag  65F9-R233E-For gaatatataccaaggtatccGAAtatgtcaactggattaag  66 F9-K230A-ForcaaatatggaatatataccGCAgtatcccggtatgtc  67 F9-K230E-ForcaaatatggaatatataccGAAgtatcccggtatgtc  68 F9-K126E-ForcctatttgcattgctgacGAAgaatacacgaacatc  69 F9-K126A-ForcctatttgcattgctgacGCAgaatacacgaacatc  70 F9-R165A-ForgttccacttgttgacGCAgccacatgtcttcgatct  71 F9-R165E-ForgttccacttgttgacGAAgccacatgtcttcgatct  72 F9-R170A-ForcgagccacatgtcttGCAtctacaaagttcacc  73 F9-R170E-ForcgagccacatgtcttGAAtctacaaagttcacc  74 F9-D[64]N-ForggcggcagttgcaagAACgacattaattcctatG 273 F9-D[64]A-ForggcggcagttgcaagGCTgacattaattcctatG 274 F9-N[157]Q-ForcctgatgtggactatgtaCAGtctactgaagctgaaacc 275 F9-N[157]D-ForcctgatgtggactatgtaGACtctactgaagctgaaacc 276 F9-N[167]Q-ForgaaaccattttggatCAGatcactcaaagcacc 277 F9-N[167]D-ForgaaaccattttggatGACatcactcaaagcacc 278 F9-S[61]A-ForccatgtttaaatggcggcGCTtgcaaggatgacattaattcc 279 F9-S[53]A-ForgatggagatcagtgtgagGCTaatccatgtttaaatggc 280 F9-T[159]A-ForgtggactatgtaaattctGCTgaagctgaaaccattttg 281 F9-T[169]A-ForCattttggataacatcGCTcaaagcacccaatcatttaatga 282 c F9-T[172]A-ForgataacatcactcaaagcGCTcaatcatttaatgac 283 F9-T[179]A-ForcaatcatttaatgacttcGCTcgggttgttggtggagaaG 284 F9-Y[155]F-ForgtttttcctgatgtggacTTCgtaaattctactgaagctG 285 F9-Y[155]H-ForgtttttcctgatgtggacCACgtaaattctactgaagctG 286 F9-Y[155]Q-ForgtttttcctgatgtggacCAGgtaaattctactgaagctG 287 F9-S[158]A-ForgtggactatgtaaatGCTactgaagctgaaacc 288 F9-S[158]D-ForgtggactatgtaaatGACactgaagctgaaacc 289 F9-S[158]E-ForgtggactatgtaaatGAGactgaagctgaaacc 290 F9-R165S-ForgttccacttgttgacAGCgccacatgtcttcgatct 291 F9-R170L-ForcgagccacatgtcttCTGtctacaaagttcacc 292 F9-K148N-ForggaagagtcttccacAACgggagatcagctttaG 293 F9-K148A-ForggaagagtcttccacGCTgggagatcagctttaG 294 F9-K148E-ForggaagagtcttccacGAGgggagatcagctttaG 295 F9-K148S-ForggaagagtcttccacAGCgggagatcagctttaG 296 F9-K148M-ForggaagagtcttccacATGgggagatcagctttaG 297 F9-E74S-ForggtgaacataatattAGCgagacagaacatacaG 298 F9-E74A-ForggtgaacataatattGCTgagacagaacatacaG 299 F9-E74R-ForggtgaacataatattAGGgagacagaacatacaG 300 F9-E74K-ForggtgaacataatattAAGgagacagaacatacaG 301 F9-H92F-For-CorrcgaattattcctcacTTCaactacaatgcaGC 302 F9-H92Y-For-CorrcgaattattcctcacTACaactacaatgcaGC 303 F9-H92E-For-CorrcgaattattcctcacGAAaactacaatgcaGC 304 F9-H92S-For-CorrcgaattattcctcacAGCaactacaatgcaGC 305 F9-T242A-ForCtggattaaggaaaaaGCTaagctcacttaagtg 306 F9-T242V-ForCtggattaaggaaaaaGTGaagctcacttaagtg 307 F9-E240N/T242A-ForgtcaactggattaagAACaaaGCTaagctcacttaagtg 308 F9-E240N/T242V-ForgtcaactggattaagAACaaaGTGaagctcacttaagtg 309 F9-E240Q-ForgtcaactggattaagCAGaaaacaaagctcacttaaG 310 F9-E240S-ForgtcaactggattaagAGCaaaacaaagctcacttaaG 311 F9-E240A-ForgtcaactggattaagGCTaaaacaaagctcacttaaG 312 F9-E240D-ForgtcaactggattaagGACaaaacaaagctcacttaaG 313 F9-N178D-ForCAaagttcaccatctatGACaacatgttctgtgctggc 314 F9-N178Y-ForCAaagttcaccatctatTACaacatgttctgtgctggc 315 F9-Y177A-ForCTacaaagttcaccatcGCTaacaacatgttctgtGC 316 F9-Y177T-ForCTacaaagttcaccatcACCaacaacatgttctgtGC 317 F9-T175R-ForcttcgatctacaaagttcAGGatctataacaacatgttc 318 F9-T175E-ForcttcgatctacaaagttcGAAatctataacaacatgttc 319 F9-T175Q-ForcttcgatctacaaagttcCAGatctataacaacatgttc 320 F9-F174I-ForGTcttcgatctacaaagATCaccatctataacaacatg 321 F9-T175R/Y177T-ForcgatctacaaagttcAGGatcACCaacaacatgttctgtG 322 F9-Y94F/K98T-ForGAattattcctcaccacaacTTCaatgcagctattaatACCt 323 acaaccatgacattGF9-F145N/K148S-For ggctggggaagagtcAACcacAGCgggagatcagctttaG 324

Table 13 below sets forth the FIX variants that were generated, with themutations indicated using numbering relative to the mature FIXpolypeptide set forth in SEQ ID NO:3, and also chymotrypsin numbering.

TABLE 13 FIX variants SEQ ID Mutation (Mature FIX Numbering) Mutation(Chymotrypsin Numbering) NO. Catalyst Biosciences WT CatalystBiosciences WT 3 N157D N[157]D 75 Y155F Y[155]F 76 A103N/N105SA[103]N/N[105]S 77 D104N/K106S D[104]N/K[106]S 78 K106N/V108SK[106]N/V[108]S 79 D85N D[85]N 80 T148A T[148]A 81 K5A K[5]A 82 D64ND[64]N 83 D64A D[64]A 84 N167D N[167]D 85 N167Q N[167]Q 86 S61A S[61]A87 S53A S[53]A 88 T159A T[159]A 89 T169A T[169]A 90 T172A T[172]A 91T179A T[179]A 92 Y155H Y[155]H 93 Y155Q Y[155]Q 94 S158A S[158]A 95S158D S[158]D 96 S158E S[158]E 97 N157Q N[157]Q 98 D203N/F205T D39N/F41T99 D85N/D203N/F205T D[85]N/D39N/F41T 100 K228N K63N 101 D85N/K228ND[85]N/K63N 102 I251S I86S 103 D85N/I251S D[85]N/I86S 104D85N/D104N/K106S/I251S D[85]N/D[104]N/K[106]S/I86S 105 A262S A95bS 106K413N K243N 107 E410N E240N 108 E239N E74N 109 T241N/H243S T76N/H78S 110K247N/N249S K82N/N84S 111 L321N L153N 112 F314N/H315S F145N/H147S 113K392N/K394S K222N/K224S 114 S319N/L321S S151N/L153S 115 N260S N95S 116Y284N Y117N 117 G317N G149N 118 R318N/A320S R150N/A152S 119 R318A R150A120 R318E R150E 121 R318Y R150Y 122 R312Q R143Q 123 R312A R143A 124R312Y R143Y 125 R312L R143L 126 V202M V38M 127 V202Y V38Y 128 D203M D39M129 D203Y D39Y 130 A204M A40M 131 A204Y A40Y 132 K400A/R403A K230A/R233A133 K400E/R403E K230E/R233E 134 R403A R233A 135 R403E R233E 136 K400AK230A 137 K400E K230E 138 K293E K126E 139 K293A K126A 140 R333A R165A141 R333E R165E 142 R338A R170A 143 R338E R170E 144 R338A/R403AR170A/R233A 145 R338E/R403E R170E/R233E 146 K293A/R403A K126A/R233A 147K293E/R403E K126E/R233E 148 K293A/R338A/R403A K126A/R170A/R233A 149K293E/R338E/R403E K126E/R170E/R233E 150 R318A/R403A R150A/R233A 151R318E/R403E R150E/R233E 152 R318Y/E410N R150Y/E240N 153 R338E/E410NR170E/E240N 154 R338E/R403E/E410N R170E/R233E/E240N 155R318Y/R338E/R403E R150Y/R170E/R233E 156 D203N/F205T/K228N D39N/F41T/K63N157 D203N/F205T/E410N D39N/F41T/E240N 158 D203N/F205T/R338ED39N/F41T/R170E 159 D203N/F205T/R338A D39N/F41T/R170A 160D203N/F205T/R318Y D39N/F41T/R150Y 161 D203N/F205T/R338E/R403ED39N/F41T/R170E/R233E 162 K228N/E410N K63N/E240N 163 K228N/R338EK63N/R170E 164 K228N/R338A K63N/R170A 165 K228N/R318Y K63N/R150Y 166K228N/R338E/R403E K63N/R170E/R233E 167 R403E/E410N R233E/E240N 168R318Y/R338E/E410N R150Y/R170E/E240N 169 K228N/R318Y/E410NK63N/R150Y/E240N 170 R318Y/R403E/E410N R150Y/R233E/E240N 171R318Y/R338E/R403E/E410N R150Y/R170E/R233E/E240N 172D203N/F205T/R318Y/E410N D39N/F41T/R150Y/E240N 173 R333S R165S 186 R338LR170L 187 K316N K148N 189 K316A K148A 190 K316E K148E 191 K316S K148S192 K316M K148M 193 E239S E74S 194 E239A E74A 195 E239R E74R 196 E239KE74K 197 H257F H92F 198 H257Y H92Y 199 H257E H92E 200 H257S H92S 201T412A T242A 202 T412V T242V 203 E410N/T412A E240N/T242A 204 E410N/T412VE240N/T242V 205 E410Q E240Q 174 E410S E240S 175 E410A E240A 176 E410DE240D 206 N346D N178D 207 N346Y N178Y 208 F314N/K316S F145N/K148S 177A103N/N105S/K228N A[103]N/N[105]S/K63N 217 D104N/K106S/K228ND[104]N/K[106]S/K63N 218 K228N/I251S K63N/I86S 180 A103N/N105S/I251SA[103]N/N[105]S/I86S 181 D104N/K106S/I251S D[104]N/K[106]S/I86S 182A103N/N105S/R318Y/R338E/R403E/ A[103]N/N[105]S/R150Y/R170E/ 219 E410NR233E/E240N D104N/K106S/R318Y/R338E/R403E/ D[104]N/K[106]S/R150Y/R170E/220 E410N R233E/E240N K228N/R318Y/R338E/R403E/E410NK63N/R150Y/R170E/R233E/E240N 221 I251S/R318Y/R338E/R403E/E410NI86S/R150Y/R170E/R233E/E240N 222 D104N/K106S/I251S/R318Y/R338E/D[104]N/K[106]S/I86S/R150Y/ 223 R403E/E410N R170E/R233E/E240ND104N/K106S/R318Y/E410N/R338E D[104]N/K[106]S/R150Y/E240N/ 224 R170EI251S/R318Y/E410N/R338E I86S/R150Y/E240N/R170E 225D104N/K106S/I251S/R318Y/R338E/ D[104]N/K[106]S/I86S/R150Y/ 226 E410N/R170E/E240N A103N/N105S/K247N/N249S A[103]N/N[105]S/K82N/N84S 178D104N/K106S/K247N/N249S D[104]N/K[106]S/K82N/N84S 179 K228N/K247N/N249SK63N/K82N/N84S 183 A103N/N105S/Y155F A[103]N/N[105]S/Y[155]F 227D104N/K106S/Y155F D[104]N/K[106]S/Y[155]F 228 Y155F/K228N Y[155]F/K63N229 Y155F/I251S Y[155]F/I86S 230 Y155F/K247N/N249S Y[155]F/K82N/N84S 231A103N/N105S/K247N/N249S/R318Y/ A[103]N/N[105]S/K82N/N84S/ 232R338E/R403E/E410N R150Y/R170E/R233E/E240N D104N/K106S/K247N/N249S/D[104]N/K[106]S/K82N/N84S/ 233 R318Y/R338E/R403E/E410NR150Y/R170E/R233E/E240N K228N/K247N/N249S/R318Y/R338E/K63N/K82N/N84S/R150Y/R170E/ 234 R403E/E410N R233E/E240NA103N/N105S/Y155F/R318Y/R338E/ A[103]N/N[105]S/Y[155]F/R150Y/ 235R403E/E410N R170E/R233E/E240N D104N/K106S/D[104]N/K[106]S/Y[155]F/R150Y/ 236 Y155F/R318Y/R338E/R403E/E410NR170E/R233E/E240N Y155F/K228N/R318Y/R338E/R403E/Y[155]F/K63N/R150Y/R170E/ 237 E410N R233E/E240NY155F/I251S/R318Y/R338E/R403E/ Y[155]F/I86S//R150Y/R170E/ 238 E410NR233E/E240N Y155F/K247N/N249S/R318Y/R338E/Y[155]F/K82N/N84S/R150Y/R170E/ 239 R403E/E410N R233E/E240NK247N/N249S/R318Y/R338E/R403E/ K82N/N84S/R150Y/R170E/R233E/ 240 E410NE240N Y155F/R318Y/R338E/R403E/E410N Y[155]F/R150Y/R170E/R233E/E240N 241K247N/N249S/R318Y/R338E/E410N K82N/N84S/R150Y/R170E/E240N 242Y155F/R318Y/R338E/E410N Y[155]F/R150Y/R170E/E240N 243Y155F/K247N/N249S/R318Y/R338E/ Y[155]F/K82N/N84S/R150Y/R170E/ 244 E410NE240N D104N/K106S/Y155F/K228N/K247N/N249S D[104]N/K[106]S/Y[155]F/ 245K63N/K82N/N84S D104N/K106S/Y155F/K247N/N249SD[104]N/K[106]S/Y[155]F/K82N/N84S 246 D104N/K106S/Y155F/K228N/D[104]N/K[106]S/Y[155]F/K63N 247 Y155F/K228N/K247N/N249SY[155]F/K63N/K82N/N84S 248 D104N/K106S/K228N/K247N/N249SD[104]N/K[106]S/K63N/K82N/N84S 184 R318Y/R338E/R403E/E410SR150Y/R170E/R233E/E240S 249 R318Y/R338E/R403E/E410N/T412VR150Y/R170E/R233E/E240N/T242V 250 R318Y/R338E/R403E/E410N/T412AR150Y/R170E/R233E/E240N/T242A 251 R318Y/R338E/R403E/T412AR150Y/R170E/R233E/T242A 252 R318Y/R338E/E410S R150Y/R170E/E240S 253R318Y/R338E/T412A R150Y/R170E/T242A 254 R318Y/R338E/E410N/T412VR150Y/R170E/E240N/T242V 255 D85N/K228N/R318Y/R338E/R403E/D[85]N/K63N/R150Y/R170E/R233E/E240N 256 E410NN260S/R318Y/R338E/R403E/E410N N95S/R150Y/R170E/R233E/E240N 257R318Y/R338E/N346D/R403E/E410N R150Y/R170E/N178D/R233E/E240N 258Y155F/N346D Y[155]F/N178D 259 Y155F/R318Y/R338E/N346D/R403E/E410NY[155]F/R150Y/R170E/N178D/ 260 R233E/E240N Y155F/N260S/N346D/Y[155]F/N95S/N178D 261 K247N/N249S/N260S K82N/N84S/N95S 262D104N/K106S/N260S D[104]N/K[106]S/N95S 185 Y155F/N260S Y[155]F/N95S 263K247N/N249S/N260S/R318Y/R338E/ K82N/N84S/N95S/R150Y/R170E/ 264R403E/E410N R233E/E240N D104N/K106S/N260S/R318Y/R338E/D[104]N/K[106]S/N95S/R150Y/ 265 R403E/E410N R170E/R233E/E240NY155F/N260S/R318Y/R338E/R403E/ Y[155]F/N95S/R150Y/R170E/ 266 E410NR233E/E240N R318Y/R338E/T343R/R403E/E410N R150Y/R170E/T175R/R233E/E240N267 R338E/T343R R170E/T175R 268 D104N/K106S/Y155F/N260SD[104]N/K[106]S/Y[155]F/N95S 269 Y155F/K247N/N249S/N260SY[155]F/K82N/N84S/N95S 270 D104N/K106S/K247N/N249S/N260SD[104]N/K[106]S/K82N/N84S/N95S 271 D104N/K106S/Y155F/K247N/N249S/N260SD[104]N/K[106]S/Y[155]F/ 272 K82N/N84S/N95S Y345A Y177A 213 Y345T Y177T214 T343R T175R 209 T343E T175E 210 T343Q T175Q 211 F342I F174I 212T343R/Y345T T175R/Y177T 215 R318Y/R338E R150Y/R170E 188Y259F/K265T/Y345T Y94F/K98T/Y177T 216 D104N/K106S/Y155F/K247N/N249S/D[104]N/K[106]S/Y[155]F/K82N/ 326 R318Y/R338E/R403E/E410NN84S/R150Y/R170E/R233E/E240N D104N/K106S/K228N/K247N/N249S/D[104]N/K[106]S/K63N/K82N/N84S/R150Y/ 327 R318Y/R338E/R403E/E410NR170E/R233E/E240N Y155F/K228N/K247N/N249S/R318Y/Y[155]F/K63N/K82N/N84S/R150Y/ 328 R338E/R403E/E410N R170E/R233E/E240NY155F/K247N/N249S/N260S/R318Y/ Y[155]F/K82N/N84S/N95S/R150Y/ 329R338E/R403E/E410N R170E/R233E/E240N Y155F/R318Y/R338E/T343R/R403E/Y[155]F/R150Y/R170E/T175R/R233E/E240N 330 E410ND104N/K106S/R318Y/R338E/T343R/ D[104]N/K[106]S/R150Y/R170E/ 331R403E/E410N T175R/R233E/E240N T343R/N346Y T175R/N178Y 332R318Y/R338E/N346Y/R403E/E410N R150Y/R170E/N178Y/R233E/E240N 333R318Y/R338E/T343R/N346Y/R403E/ R150Y/R170E/T175R/N178Y/R233E/ 334 E410NE240N T343R/N346D T175R/N178D 335 R318Y/R338E/T343R/N346D/R403E/R150Y/R170E/T175R/N178D/R233E/ 336 E410N E240NR318Y/R338E/Y345A/R403E/E410N R150Y/R170E/Y177A/R233E/E240N 337R318Y/R338E/Y345A/N346D/R403E/ R150Y/R170E/Y177A/N178D/R233E/E240N 338E410N Y155F/K247N/N249S/R318Y/R338E/ Y[155]F/K82N/N84S/R150Y/R170E/ 339R403E R233E K247N/N249S/R318Y/R338E/R403E K82N/N84S/R150Y/R170E/R233E340 Y155F/K247N/N249S/R318Y/R403E/ Y[155]F/K82N/N84S/R150Y/R233E/ 341E410N E240N K247N/N249S/R318Y/R403E/E410N K82N/N84S/R150Y/R233E/E240N342 Y155F/K247N/N249S/R338E/R403E/ Y[155]F/K82N/N84S/R170E/R233E/ 343E410N E240N K247N/N249S/R338E/R403E/E410N K82N/N84S/R170E/R233E/E240N344 R318Y/R338E/T343R/R403E R150Y/R170E/T175R/R233E 345Y155F/R318Y/R338E/T343R/R403E Y[155]F/R150Y/R170E/T175R/R233E 346R318Y/R338E/T343R/E410N R150Y/R170E/T175R/E240N 347Y155F/R318Y/R338E/T343R/E410N Y[155]F/R150Y/R170E/T175R/E240N 348R318Y/T343R/R403E/E410N R150Y/T175R/R233E/E240N 349Y155F/R318Y/T343R/R403E/E410N Y[155]F/R150Y/T175R/R233E/E240N 350R338E/T343R/R403E/E410N R170E/T175R/R233E/E240N 351Y155F/R338E/T343R/R403E/E410N Y[155]F/R170E/T175R/R233E/E240N 352Y155F/K247N/N249S/R318Y/R338E/ Y[155]F/K82N/N84S/R150Y/R170E/ 353T343R/R403E/E410N T175R/R233E/E240N K247N/N249S/R318Y/R338E/T343R/K82N/N84S/R150Y/R170E/T175R/ 354 R403E/E410N R233E/E240NK228N/I251S/R318Y/R338E/R403E/ K63N/I86S/R150Y/R170E/R233E/ 355 E410NE240N Y155F/K228N/I251S/R318Y/R338E/ Y[155]F/K63N/I86S/R150Y/R170E/ 356R403E/E410N R233E/E240N N260S/R318Y/R338E/T343R/R403E/N95S/R150Y/R170E/T175R/R233E/ 357 E410N E240NY155F/N260S/R318Y/R338E/T343R/ Y[155]F/N95S/R150Y/R170E/T175R/R233E/ 358R403E/E410N E240N K228N/K247N/N249S/R318Y/R338E/K63N/K82N/N84S/R150Y/R170E/ 359 T343R/R403E/E410N T175R/R233E/E240NY155F/K228N/K247N/N249S/R318Y/ Y[155]F/K63N/K82N/N84S/R150Y/ 360R338E/T343R/R403E/E410N R170E/T175R/R233E/E240N Y155F/R338E/T343R/R403EY[155]F/R170E/T175R/R233E 361 R338E/T343R/R403E R170E/T175R/R233E 362Y155F/R338E/T343R/R403E/E410S Y[155]F/R170E/T175R/R233E/E240S 363Y155F/N260S/R338E/T343R/R403E Y[155]F/N95S/R170E/T175R/R233E 364Y155F/I251S/R338E/T343R/R403E Y[155]F/I86S/R170E/T175R/R233E 365R318Y/R338E/T343R/R403E/E410S R150Y/R170E/T175R/R233E/E240S 366Y155F/K247N/N249S/T343R/R403E Y[155]F/K82N/N84S/T175R/R233E 367Y155F/K247N/N249S/R318Y/R338E/ Y[155]F/K82N/N84S/R150Y/R170E/ 368T343R/R403E T175R/R233E K247N/N249S/R318Y/R338E/T343R/K82N/N84S/R150Y/R170E/T175R/ 369 R403E R233EY155F/K247N/N249S/R338E/T343R/ Y[155]F/K82N/N84S/R170E/T175R/ 370R403E/E410N R233E/E240N K247N/N249S/R338E/T343R/R403E/K82N/N84S/R170E/T175R/R233E/ 371 E410N E240NY155F/K247N/N249S/R318Y/R338E Y[155]F/K82N/N84S/R150Y/R170E 372Y155F/K247N/N249S/R318Y/T343R Y[155]F/K82N/N84S/R150Y/T175R 373Y155F/K247N/N249S/R318Y/R403E Y[155]F/K82N/N84S/R150Y/R233E 374Y155F/K247N/N249S/R318Y/E410N Y[155]F/K82N/N84S/R150Y/E240N 375Y155F/K247N/N249S/R338E/R403E Y[155]F/K82N/N84S/R170E/R233E 376Y155F/K247N/N249S/R338E/T343R Y[155]F/K82N/N84S/R170E/T175R 377Y155F/K247N/N249S/R318Y/R338E/ Y[155]F/K82N/N84S/R150Y/R170E/ 378T343R/E410N T175R/E240N K247N/N249S/R318Y/R338E/T343R/K82N/N84S/R150Y/R170E/T175R/ 379 E410N E240NY155F/K247N/N249S/R318Y/T343R/ Y[155]F/K82N/N84S/R150Y/T175R/ 380R403E/E410N R233E/E240N K247N/N249S/R318Y/T343R/R403E/K82N/N84S/R150Y/T175R/R233E/ 381 E410N E240NY155F/K247N/N249S/R338E/E410N Y[155]F/K82N/N84S/R170E/E240N 382Y155F/K247N/N249S/R318Y/T343R/ Y[155]F/K82N/N84S/R150Y/T175R/ 383 R403ER233E K247N/N249S/R318Y/T343R/R403E K82N/N84S/R150Y/T175R/R233E 384Y155F/K247N/N249S/R318Y/T343R/ Y[155]F/K82N/N84S/R150Y/T175R/ 385 E410NE240N K247N/N249S/R318Y/T343R/E410N K82N/N84S/R150Y/T175R/E240N 386Y155F/K247N/N249S/R338E/T343R/ Y[155]F/K82N/N84S/R170E/T175R/ 387 R403ER233E K247N/N249S/R338E/T343R/R403E K82N/N84S/R170E/T175R/R233E 388Y155F/K247N/N249S/R338E/T343R/ Y[155]F/K82N/N84S/R170E/T175R/ 389 E410NE240N K247N/N249S/R338E/T343R/E410N K82N/N84S/R170E/T175R/E240N 390Y155F/K247N/N249S/T343R/R403E/ Y[155]F/K82N/N84S/T175R/R233E/ 391 E410NE240N K247N/N249S/T343R/R403E/E410N K82N/N84S/T175R/R233E/E240N 392Y155F/R318Y/R338E/T343R Y[155]F/R150Y/R170E/T175R 393 R318Y/R338E/T343RR150Y/R170E/T175R 394 Y155F/R318Y/T343R/R403E Y[155]F/R150Y/T175R/R233E395 Y155F/T343R/R403E/E410N Y[155]F/T175R/R233E/E240N 396Y155F/K247N/N249S/R318Y/R338E/ Y[155]F/K82N/N84S/R150Y/R170E/ 397 T343RT175R K247N/N249S/R318Y/R338E/T343R K82N/N84S/R150Y/R170E/T175R 398Y155F/K247N/N249S/T343R/E410N Y[155]F/K82N/N84S/T175R/E240N 399Y155F/K247N/N249S/R403E/E410N Y[155]F/K82N/N84S/R233E/E240N 400Y155F/R338E/T343R/E410N Y[155]F/R170E/T175R/E240N 401 R338E/T343R/E410NR170E/T175R/E240N 402 Y155F/R318Y/T343R/E410N Y[155]F/R150Y/T175R/E240N403 R318Y/T343R/E410N R150Y/T175R/E240N 404K228N/R318Y/R338E/T343R/R403E/ K63N/R150Y/R170E/T175R/R233E/ 405 E410NE240N K228N/K247N/N249S/R318Y/R338E/ K63N/K82N/N84S/R150Y/R170E/ 406T343R/R403E T175R/R233E K228N/K247N/N249S/R318Y/R338E/K63N/K82N/N84S/R150Y/R170E/ 407 T343R/E410N T175R/E240NK228N/K247N/N249S/R318Y/T343R/ K63N/K82N/N84S/R150Y/T175R/ 408R403E/E410N R233E/E240N Y155F/R338E/R403E/E410NY[155]F/R170E/R233E/E240N 409 Y155F/R318Y/R338E/R403EY[155]F/R150Y/R170E/R233E 410 Y155F/R318Y/R403E/E410NY[155]F/R150Y/R233E/E240N 411 Y1N Y[1]N 412

C. Expression and Purification of FIX Polypeptides

Wild type and variant FIX polypeptides were expressed in CHO-Express(CHOX) cells (Excellgene). CHO Express (CHOX) cells were maintained inDM204B Complete medium (Irvine Scientific) and used to inoculateproduction seed cultures. Seed cultures were grown in the same media toapproximately 1.4×10⁷ viable cells (vc)/mL and approximately 100 mL usedto inoculate approximately 1.0 L of DM204B Complete media, so that theinoculation density was 1.2×10⁶ vc/mL. This culture was grown for 3 daysto reach 13-16×10⁶ vc/mL on the day of transfection. A transfectioncomplex was formed by mixing FIX plasmid DNA (3.2 mg) withPolyethylenimine “MAX” (PEI—20.5 mg (Polysciences)) and diluting to 1.0L with serum-free TfMAX2 transfection medium (Mediatech). This mixturewas then added to the 1.0 L production culture. 1.0 L aliquots of thecells plus transfection mix were split into 2×3 L baffled FernbackFlasks and allowed to express for 4 days before harvesting the crudeFIX. Culture supernatants were then harvested by filtration and FIX waspurified.

Larger-scale cultures of 10 L or greater were produced in WAVEbioreactors (GE Healthcare). 20 L wave bags were inoculated withapproximately 400 mL of seed culture, grown as described above, with 4.6L of DM204B Complete media to a seeding density of 1.2×10⁶ vc/mL. TheWAVE bioreactor was set to a rocking angle of 6 degrees, rocking rate of24 rpm at 37.1° C. in order to allow the cells to reach a cell densityof 13-16×10⁶ vc/mL 3 days later. 16 mg of FIX plasmid DNA and 102.5 mgof PEI were combined to form a transfection complex, which was dilutedin 5.0 L of TfMAX2 prior to addition to the culture on the WAVEbioreactor, 3 days after the initial seeding. While the Transfectioncomplex plus TfMAX media was added to the wave bag, the rocking angle ofthe WAVE Bioreactor was set to 8 degrees and the temperature to 33° C.,while the other settings remained the same. The culture was allowed toexpress for 4 days before harvesting the crude FIX. The contents of thewave bags were allowed to settle for 3 hrs at 4° C. prior to harvestingthe culture supernatant through a CUNO depth filter and then the FIX waspurified.

FIX polypeptides were purified using a Capto Q column (GE Healthcare),to which FIX polypeptides with functional Gla domains adsorb, followedby a calcium elution step. Typically, EDTA (10 mM), Tris (25 mM, pH8.0), and Tween-80 (0.001%) were added to the culture supernatant fromthe transfected cells. The samples were loaded onto a Capto Q columnthat had been pre-equilibrated with Buffer B (25 mM Tris pH 8, 1 M NaCl,0.001% Tween-80), followed by equilibration with Buffer A (25 mM Tris pH8, 0.15 M NaCl, 0.001% Tween-80). Immediately following completion ofsample loading, the column was washed with 14% Buffer B (86% Buffer A)for 20 column volumes. Buffer C (25 mM Tris pH 8, 0.2 M NaCl, 0.001%Tween-80, 10 mM CaCl₂) was then applied to the column to elute the FIXpolypeptides that were collected as a pool.

The eluted pool was further purified using a Q Sepharose HP column (GEHealthcare). The sample was prepared for application by diluting with 2volumes of Buffer D (25 mM Tris pH 8, 0.001% Tween-80). The dilutedsample was loaded onto a Q Sepharose HP column that had beenpre-equilibrated with Buffer F (25 mM Tris pH 8, 1 M NaCl, 2.5 mM CaCl₂,0.001% Tween-80), followed by Buffer E (25 mM Tris pH 8, 2.5 mM CaCl₂,0.001% Tween-80). After washing with 4% Buffer F (96% Buffer E), agradient from 4-40% Buffer F was applied to the column and fractionswere collected. Fractions containing FIX polypeptides were then pooled.

D. Purification to Enrich for Glycosylated Polypeptides.

The extent of glycosylation of the modified FIX polypeptides wasestimated using SDS-polyacrylamide gel electrophoresis.Hyperglycosylation was assessed by comparison of the migration patternof the modified FIX polypeptide with a wild type FIX, Benefix®Coagulation FIX. Hyperglycosylated forms of the enzyme migrated slower,exhibiting a higher apparent molecular weight, than the wild typepolypeptide. It was observed that the polypeptides containing the E240Nmutation, which introduces a non-native N-glycosylation site at position240, were only partially glycosylated (approximately 20% glycosylation).To enrich for the hyperglycosylated form, a modification of thepurification process described above was performed.

The first step of purification was performed using the Capto Q column,as described above. The eluted pool from this column was diluted with 2volumes of Buffer D (as above) and the sample was loaded onto a HeparinSepharose column that had been pre-equilibrated with Buffer F (asabove), followed by Buffer E (as above). The column was then developedwith a gradient from 0% to 70% Buffer F and fractions were collected.The hyperglycosylated form of the E410N variant eluted from the columnin approximately 35% Buffer F, whereas the non-hyperglycosylated formeluted in approximately 50% Buffer F. Each collected pool was furtherpurified on the Q Sepharose HP column as described above. By this methoda pool containing approximately 80% hyperglycosylated form of the E410Nvariant was obtained. The extent of hyperglycosylation was estimated byvisual inspection of SDS-polyacrylamide gel electrophoresis.

Example 2 Activation of FX and Determination of the Catalytically ActiveProtease (FXa) Concentration Using the Active Site TitrantFluorescein-Mono-p′-Guanidinobenzoate (FMGB)

The concentration of Factor X (FX) in a stock of FX that can becomecatalytically active was determined. This stock of FX was then used insubsequent studies to calculate the catalytic activity of FIX variantsfor FX. Following activation of FX to FXa, the active site titrationassay was carried out essentially as described by Bock et al. (Archivesof Biochemistry and Biophysics (1989) 273:375-388) using the fluorogenicester substrate fluorescein-mono-p′-guanidinobenzoate (FMGB), with a fewminor modifications. FMGB readily reacts with FXa, but not FX orinactive protease, to form an effectively stable acyl-enzymeintermediate under conditions in which the concentration of FMGB issaturating and deacylation is especially slow and rate limiting forcatalysis. Under these conditions, the FXa protease undergoes a singlecatalytic turnover to release the fluorescein fluorophore. When theinitial burst of fluorescence is calibrated to an external concentrationstandard curve of fluorescein fluorescence, the concentration of activesites can be calculated.

A. Activation of FX to FXa

The concentration of FX in a stock solution that is able to becomecatalytically active was determined by activation of FX samples withRussell's Viper Venom, followed by titrating the active FX (FXa) withFMGB. FX zymogen stocks were first pre-treated by the supplier with DFP(diisopropylfluorophosphate) and EGR-cmk to reduce the background FXaactivity. FXa activation reactions were prepared with a finalconcentration of 10 μM FX (based on the A₂₈₀ absorbance and anextinction coefficient of 1.16) in a final volume of 50-100 μL in areaction buffer containing 100 mM Tris, 50 mM NaCl, 5 mM CaCl₂, 0.1% PEG8000, pH 8.1. Activation was initiated by the addition of Russell'sViper Venom (RVV-Xase; Heamatologic Technologies, Inc.) to a finalconcentration of 5 μg/mL (5 μL of a 98 μg/mL dilution per 100 μLreaction or 2.5 μL per 50 μL reaction) at 37° C. for 45-60 min ofactivation time (previously determined to represent complete activationby collecting samples every 15 min and testing the increase in cleavageof Spectrafluor FXa fluorogenic substrate). Reactions were quenched with1/10 volume of quench buffer containing 100 mM Tris, 50 mM NaCl, 5 mM,100 mM EDTA, 0.1% PEG 8000, pH 8.1.

B. Active Site Titration

The active site titration assays were performed with a 1 mL reactionvolume in a 0.4 cm×1 cm quartz cuvette under continuous stirring.Reactions contained 100-400 nM of the freshly activated FXa and 5 μMFMGB in an assay buffer containing 30 mM Hepes, 135 mM NaCl, 1 mM EDTAand 0.1% PEG 8000, pH 7.4. FMGB was prepared at a stock concentration of0.01 M in DMF based on the dry weight and the concentration confirmed byabsorbance spectroscopy at 452 nm using an extinction coefficient of19,498 M⁻¹ cm⁻¹ in Phosphate Buffered Saline (PBS), pH 7.2. Assays wereinitiated by adding 5 μL of 1 mM FMGB (5 μM final concentration) to 1 mLof 1× assay buffer and first measuring the background hydrolysis of FMGBfor ˜150-200 seconds before the addition of FXa to a final concentrationof ˜100-400 nM. The release of fluorescein fluorescence in the burstphase of the reaction was followed for an additional 3600 seconds.

The amount of fluorescein released following catalysis of FMGB by FXawas determined using a standard curve of free fluorescein. Thefluorescein standard solution was freshly prepared at a stockconcentration of ˜70-150 mM in DMF and the accurate concentration wasconfirmed by absorbance spectroscopy under standard conditions at 496 nmusing an extinction coefficient of 89,125 M⁻¹ cm⁻¹ in 0.1 N NaOH. Astandard curve of free fluorescein was then prepared by titration of theabsorbance-calibrated fluorescein standard into 1× assay buffer in 20 nMsteps to a final concentration of 260-300 nM.

For data analysis, reaction traces were imported into the Graphpad Prismsoftware package and the contribution of background hydrolysis wassubtracted from the curve by extrapolation of the initial measured rateof spontaneous FMGB hydrolysis, which was typically less than 5% of thetotal fluorescence burst. The corrected curve was fit to a singleexponential equation with a linear component (to account for the slowrate of deacylation) of the form ΔFluorescence=Amp(1-e^(−kt))+Bt, whereAmp=the amplitude of the burst phase under the saturating assayconditions outline above, k is the observed first order rate constantfor acyl-enzyme formation and B is a bulk rate constant associated withcomplete turnover of FMGB. The concentration of active FXa protease wascalculated by comparison of the fit parameter for amplitude to thefluorescein standard curve. The values from multiple assays weremeasured, averaged and the standard deviation determined. The amount ofactive FXa in the preparation directly represents the concentration ofFX in a stock preparation that can be activated by FIXa. This activesite titrated value was employed when calculating the concentration ofFX to be used in an indirect assay, such as the cofactor-dependent assaydescribed in Example 4, below.

Example 3 Activation of FIX and Determination of the CatalyticallyActive Protease (FIXa) Concentration Using the Active Site Titrant4-Methylumbelliferyl p′-Guanidinobenzoate (MUGB)

The concentration of Factor IX (FIX) in a stock solution of the FIXzymogen that is able to become catalytically active was determined byactivation of FIX samples, including FIX variants, with Factor XIa(FXIa; Heamatologic Technologies, Inc.) followed by titrating the activeFactor IX (FIXa) with 4-methylumbelliferyl p′-guanidinobenzoate (MUGB).

A. Activation of FIX to FIXa

Total protein concentrations in the FIX polypeptide preparations weredetermined by the A₂₈₀ absorbance using an extinction coefficient uniquefor each variant (i.e. ε₂₈₀=number of Tyr residues×1490+number Trpresidues×5500+number Cys residues×125). Activation reactions of FIX toFIXa were prepared at a final concentration of 10 μM FIX in a finalvolume of 200-500 μL in a reaction buffer containing 100 mM Tris, 50 mMNaCl, 5 mM CaCl₂, 0.1% PEG 8000, pH 8.1. Activations were initiated bythe addition of FXIa or biotinylated FXIa to a final concentration of 20nM at 37° C. for 60 min of activation time. A 60 minute activation timewas previously determined to represent complete activation by collectingsamples every 15 min and assaying for total cleavage by SDS-PAGE.

The free FXIa or biotinylated FXIa used in the activation reaction wasthen removed from the samples using one of two methods that produceequivalent results, each removing greater than 95-97% of the catalyticFXIa. In the first method, which was used to remove free FXIa,activation reactions initiated with FXIa were mixed with an anti-FXIamonoclonal antibody (Abcam 20377) to a final concentration of 50 nM for60 min at 37° C. Antibody capture of free FXIa was followed by theaddition of washed protein G Dynal Beads (30 mg/mL; Invitrogen) to afinal concentration of 25% vol:vol for an additional 120 min at roomtemperature. The Dynal Beads were removed from the solution per themanufacturer's instructions. In the second method, which was used toremoved biotinylated FXIa, activation reactions using biotinylated FXIawere mixed with Streptavidin Dynal Beads (10 mg/mL; Invitrogen) to afinal concentration of 10% vol:vol for 60 min at room temperature. TheDynal Beads were then removed per the manufacturer's instructions.Following removal of the FXIa, the total protein concentrations ofactivated FIXa samples were determined by A_(lso) absorbance using anextinction coefficient unique for each variant (as described above).

B. Active Site Titration of FIXa

The concentration of catalytically active FIXa in an activated stocksolution was determined by titrating the FIXa samples with a fluorogenicester substrate, 4-methylumbelliferyl p′-guanidinobenzoate (MUGB). Theprinciple titration assay was carried out essentially as described byPayne et al. (Biochemistry (1996) 35:7100-7106) with a few minormodifications to account for the slower reactivity of MUGB with FIXa.MUGB readily reacts with FIXa, but not FIX or inactive protease, to forman effectively stable acyl-enzyme intermediate under conditions in whichthe concentration of MUGB is saturating and deacylation is especiallyslow and rate limiting for catalysis. Under these conditions, the FIXaprotease undergoes a single catalytic turnover to release the4-methylumbelliferone fluorophore (4-MU). When the initial burst offluorescence is calibrated to an external concentration standard curveof 4-MU fluorescence, the concentration of active sites can becalculated.

Assays were performed with a 1 mL reaction volume in a 0.4 cm×1 cmquartz cuvette, under continuous stirring with an assay buffercontaining 50 mM Hepes, 100 mM NaCl, 5 mM CaCl₂ and 0.1% PEG 8000, pH7.6. MUGB was prepared at a stock concentration of 0.04 M in DMSO basedon the dry weight and diluted to a working concentration of 2 mM inDMSO. Titration assays were initiated by adding 4 μL of 2 mM MUGB to afinal concentration of 8 μM in 1× assay buffer and first measuring thebackground hydrolysis of MUGB for ˜200-300 seconds before the additionof the FIXa or FIXa variant to a final concentration of 100-200 nM basedon the total protein concentration determined for the activationreaction after removal of FXIa. The release of 4-MU fluorescence in theburst phase of the reaction was followed for a total of 2 hours in orderto acquire sufficient data from the initial burst and subsequent steadystate phases.

The amount of 4-MU released following catalysis of MUGB by FIXa wasdetermined using a standard curve of 4-MU. A 4-MU standard solution wasprepared at a stock concentration of 0.5 M in DMSO and the concentrationconfirmed by absorbance spectroscopy at 360 nm using an extinctioncoefficient of 19,000 M⁻¹ cm⁻¹ in 50 mM Tris buffer, pH 9.0. Thestandard curve of free 4-MU was prepared by titration of theabsorbance-calibrated 4-MU into 1× assay buffer in 20 nM steps to afinal concentration of 260-300 nM 4-MU.

For data analysis, reaction traces were imported into the Graphpad Prismsoftware package and the contribution of background hydrolysis wassubtracted from the curve by extrapolation of the initial measured rateof spontaneous MUGB hydrolysis, which was typically less than 5% of thetotal fluorescence burst. The corrected curve was fit to a singleexponential equation with a linear component (to account for the slowrate of deacylation in the steady state phase) of the formΔFluorescence=Amp(1-e^(−kt))+Bt, where Amp=the amplitude of the burstphase under the saturating assay conditions outline above, k is theobserved first order rate constant for acyl-enzyme formation and B is abulk rate constant associated with complete turnover of MUGB. Theconcentration of active FIXa protease is calculated by comparison of thefit parameter for amplitude to the 4-MU standard curve. The values frommultiple assays were measured, averaged and the standard deviationdetermined. The concentration of FIX zymogen, which may becomeactivated, in a stock solution was then determined by multiplying theA₂₈₀ determined total concentration of the FIX zymogen by theexperimentally determined fraction active value for the fully activatedsample (concentration of active FIXa/total concentration of FIXa).

Example 4 Determination of the Catalytic Activity of FIXa for itsSubstrate, Factor X

The catalytic activity of the FIXa variants for the substrate, Factor X(FX), was assessed indirectly in a fluorogenic assay by assaying for theactivity of FXa, generated upon activation by FIXa, on the syntheticsubstrate Spectrafluor FXa. A range of FX concentrations were used tocalculate the kinetic rate constants where the substrate protease (FX)was in excess by at least a 1000-fold over the concentration of theactivating protease (FIXa). Briefly, activated and active site titratedFIXa was incubated in a calcium containing buffer with recombinantFVIII, phospholipid vesicles and alpha-thrombin (to activate FVIII toFVIIIa), forming the tenase (Xase) complex. The activity ofalpha-thrombin was then quenched by the addition of a highly specificthrombin inhibitor, hirudin, prior to initiating the assay. FIXavariants (as part of the Xase complex) were subsequently mixed withvarious concentrations of FX and the fluorescent substrate, SpectrafluorFXa (CH₃SO₂-D-CHA-Gly-Arg-AMC) to initiate the assay. The release of thefree fluorophore, AMC (7-amino-4-methylcoumarin) following catalysis ofSpectrafluor FXa by FXa was then assessed continuously over a timeperiod, and the kinetic rate constants of the FIXa variants determined.

A. Assay Protocol

For assays evaluating the kinetic rate of FX activation by FIXa in thepresence of FVIIIa and phospholipids, recombinant FVIII (Kogenate FS®;Bayer healthcare) was first resuspended in 5 mL of the provided diluentaccording to the manufacturer's instructions. The molar concentration ofFVIII was then determined by absorbance at 280 nm using an extinctioncoefficient of 1.567 mg⁻¹ mL cm⁻¹ and a molecular weight of 163.6 kDa.The FIX variants were expressed, purified, activated and active sitetitrated as described in Examples 1-3, above. FIXa variants were thenserially diluted to a concentration of 16 pM in a 200 μL volume of 1×Buffer A (20 mM Hepes/150 mM NaCl/5 mM CaCl₂/0.1% BSA/0.1% PEG-8000, pH7.4). In preparation for activation of FVIII to FVIIIa in the presenceof FIXa and phospholipids, alpha-thrombin (Heamatologic Technologies,Inc.) and hirudin (American Diagnostica) were each diluted in a 1.0 mLvolume of 1× Buffer A to 64 nM and 640 nM, respectively. ReconstitutedFVIII was further diluted to a concentration of 267 nM in a 10 mL volumeof 1× Buffer A containing 267 μM freshly resuspended phospholipids (75%phosphatidylcholine (PC)/25% phospatidylserine (PS); PS/PC vesicles ˜120nm in diameter; Avanti Polar Lipids). FVIII was activated to FVIIIa bymixing 600 μL of the above FVIII/PC/PS solution with 100 μL of the 16 pMwild-type FIXa or FIXa variant dilution and 50 μL of the 64 nMalpha-thrombin solution followed by 15 minutes of incubation at 25° C.Activation reactions were subsequently quenched by the addition of 50 μLof the above 640 nM hirudin solution for 5 min at 25° C. prior toinitiating the kinetic assay for FX activation. The final concentrationof reagents in the 800 μL Xase complex solutions was as follows: 2 pMFIXa variant, 200 nM FVIIIa, 200 μM PC/PS vesicles, 4 nM alpha-thrombin(inhibited) and 40 nM hirudin.

A total of 25 μL of each Xase complex solution(FIXa/FVIIIa/Phospholipids/Ca²⁺) was aliquoted into a 96-well half-areablack assay plate according to a predefined plate map (4 FIXavariants/plate). A solution of 900 nM active site titrated andDFP/EGR-cmk treated FX (see Example 2, above) was prepared in 5.6 mL of1× Buffer A containing 1.0 mM Spectrafluor Xa substrate. Thisrepresented the highest concentration of FX tested and a sufficientvolume for 4 assays. The FX/Spectrafluor Xa solution was then seriallydiluted 1.8-fold in an 8-channel deep-well polypropylene plate with afinal volume of 2.5 mL 1× Buffer A that contains 1.0 mM Spectrafluor Xa,resulting in final dilutions of 900 nM, 500 nM, 277.8 nM, 154.3 nM, 85.7nM, 47.6 nM, 25.6 nM and 0 nM FX. Alternatively in some assays, the theFX/Specrafluor Xa solution was then serially diluted 1.5-fold in a12-channel deep-well polypropylene plate with a final volume of 2.5 mL1× Buffer A that contains 1.0 mM Spectrafluor Xa, resulting in finaldilutions of 900 nM, 600 nM, 400 nM, 266.7 nM, 177.8 nM, 118.5 nM, 79.0nM, 52.7 nM, 35.1 nM, 23.4 nM, 15.6 nM and 0 nM FX. Assay reactions weretypically initiated using a BioMek FX liquid handling system programmedto dispense 25 μL of the FX/Spectrafluor Xa dilutions into 4 assayplates containing 25 μL of each FIXa variant (Xase complex). The finalconcentrations of the reagents in the assay were as follows: 1 pM FIXa,100 nM FVIIIa, 100 μM PC/PS vesicles, 0.5 mM Spectrafluor Xa, 2 nMalpha-thrombin (inhibited), 20 nM hirudin and FX dilutions of 0 nM to450 nM. Reactions were monitored in a SpectraMax fluorescence platereader for 30 min at 37° C. A standard curve of free AMC served as theconversion factor for RFU to μM in the subsequent data analysiscalculations using a dose range that covered 0 μM to 100 μM AMC.

B. Data Analysis

All equations used to determine the steady-state kinetics of thecatalysis of FX by FIXa are based on those described in the reference“Zymogen-Activation Kinetics: Modulatory effects oftrans-4-(aminomethyl)cyclohexane-1-carboxylic acid and poly-D-lysine onplasminogen activation” in Petersen, et al. (1985) Biochem. J.225:149-158. The theory for the steady-state kinetics of the systemdescribed by Scheme A (see below) is described by the expression ofequation (1) that represents a parabolic accumulation of product.

According to the mechanism of Scheme A, a₀ is the concentration ofactivating protease (FIXa), z₀ is the concentration of zymogen (FX),k_(a) and K_(z) represent the k_(cat) and K_(M) for theactivator-catalyzed conversion of zymogen to active enzyme (FXa),whereas k_(e) and K_(s) represent the k_(cat) and K_(M) for conversionof substrate to product by FXa over a given time t:

$\begin{matrix}{p = {a_{0}\frac{k_{a}\left\lbrack z_{0} \right\rbrack}{K_{z} + \left\lbrack z_{0} \right\rbrack}*\frac{k_{e}\left\lbrack S_{0} \right\rbrack}{K_{s} + \left\lbrack S_{0} \right\rbrack}*\frac{t^{2}}{2}}} & {{Equation}\mspace{14mu} (1)}\end{matrix}$

For analysis of progress curves, equation (1) was re-cast in the form ofequation (2) where the steady-state kinetics of FXa hydrolysis of thefluorogenic substrate were determined independently and replaced by thecompound constant k₂.

$\begin{matrix}{p = {a_{0}\frac{k_{a}\left\lbrack z_{0} \right\rbrack}{K_{z} + \left\lbrack z_{0} \right\rbrack}*k_{2}*\frac{t^{2}}{2}}} & {{Equation}\mspace{14mu} (2)}\end{matrix}$

The FXa activity on Spectrofluor FXa in 1× Buffer A was independentlydetermined to have a K_(M) of 313.0 μM and a k_(cat) value of 146.4 s⁻¹.Substitution of these values into equation (3) gave a k₂ correctionfactor of 90 s⁻¹.

$\begin{matrix}{k_{2} = \frac{k_{e}\left\lbrack S_{0} \right\rbrack}{K_{M} + \left\lbrack S_{0} \right\rbrack}} & {{Equation}\mspace{14mu} (3)}\end{matrix}$

To determine the degree of FIXa catalytic activity, raw data collectedwith the SoftMax Pro application (Molecular Devices) were exported as.XML files or .TXT files. Further non-linear data analyses wereperformed with XLfit4, a software package for automated curve fittingand statistical analysis within the Microsoft Excel spreadsheetenvironment (IDBS Software) or directly within the ActivityBase softwarepackage using the XE Runner data analysis module (IDBS Software). Thespreadsheet template was set up to automatically fit the parabolicreaction velocities (μM/sec²) of the tested FIXa variants at each FXconcentration to the function of a standard rectangular hyperbola (i.e.Michaelis Menten equation) given by equation (4) to yield the fit valuesfor V_(max) and K_(M).

$\begin{matrix}{{{Reaction}\mspace{14mu} {{Velocity}\left( {{µM}\text{/}\sec^{2}} \right)}} = \frac{V_{\max}\left\lbrack S_{0} \right\rbrack}{K_{M} + \left\lbrack S_{0} \right\rbrack}} & {{Equation}\mspace{14mu} (4)}\end{matrix}$

The k_(cat) value for the tested FIXa variant was then calculated fromthe fit value for V_(max) (μM/sec²) by equation (5).

$\begin{matrix}{k_{cat} = \frac{V_{{ma}x}}{\lbrack{FIXa}\rbrack*0.5*k_{2}}} & {{Equation}\mspace{14mu} (5)}\end{matrix}$

The specificity constant k_(cat)/K_(M) was calculated directly from thefit value of K_(M) and the calculated k_(cat) that arose from evaluationof equation (5) above.

Tables 14-19 set forth the catalytic activity for each of the FIXavariants assayed. Also assayed were recombinant wild-type FIXa (termedCatalyst Biosciences WT; generated as described above in Example 1),plasma purified FIXa (Haematologic Technologies, Inc.), and BeneFIX®(Coagulation Factor IX (Recombinant); Wyeth). Tables 14-15 present theresults expressed as the kinetic constant for catalytic activity,k_(cat)/K_(M) (M⁻¹s⁻¹), and also as the percentage of the activity ofthe wild-type FIXa, wherein the activity is catalytic activity,k_(cat)/K_(M) (M⁻¹s⁻¹) of each FIXa variant for its substrate, FX. Theindividual rate constants k_(cat) and K_(M) are provided in Tables 16-17and 18-19, respectively. Tables 15, 17 and 19 reflect data foradditional FIXa variants and provide new overall averages calculated toinclude additional experimental replicates (n) for FIXa variants inTables 14, 16 and 18. Where the activity of the FIXa variant wascompared to wild-type FIXa, it was compared to a recombinant wild-typeFIXa polypeptide that was expressed and purified using the sameconditions as used for the variant FIXa polypeptides to ensure that anydifferences in activity were the result of the mutation(s), and not theresult of differences in, for example, post-translational modificationsassociated with different expression systems. Thus, the wild-type FIXapolypeptide used for comparison was the recombinant wild-type FIXagenerated from cloning the FIX gene set forth in SEQ ID NO:1 andexpressed from CHOX cells as a polypeptide with an amino acid sequenceset forth in SEQ ID NO:3, as described in Example 1 (i.e. CatalystBiosciences WT FIX polypeptide). The standard deviation (S.D.),coefficient of variation (as a percentage; % CV) and the number ofassays performed (n) also are provided for each kinetic parameter.

The observed catalytic activities of the FIXa variants ranged from nodetectable Xase activity in a few variants (e.g. FIXa-F314N/H315S,FIXa-G317N, FIXa-R318N/A320S and FIXa-K400E/R403E) to a greater than10-fold increase in k_(cat)/K_(M) for the activation of FX compared towild-type FIXa. Some of the variants displayed markedly increasedcatalytic activity compared to the wild-type FIXa, including FIXa-R338E,FIXa-R338A, FIXa-T343R, FIXa-E410N and combinations thereof such asFIXa-R318Y/R338E/E410N, FIXa-R318Y/R338E/R402E/E410N,FIXa-R318Y/R338E/T343R/R402E/E410N, FIXa-R318Y/R338E/T343R/E410N andFIXa-R338E/T343R displayed some of the greatest increases in catalyticactivity. Although several FIXa variants with single or multipleadditional glycosylation sites demonstrated close to wild type activity(e.g. FIXa-I251S, FIXa-D85N/I251S, FIXa-K63N, FIXa-K247N/N249S andFIXa-K63N/K247N/N249S) or improved activity when combined with othermutations (e.g. FIXa-K247N/N249S/R338E/T343R/R403E andFIXa-K247N/N249S/R318Y/R338E/T343R/R403E/E410N), others showed reducedcatalytic activity. The augmented catalytic activity was due toimprovements in k_(cat) or K_(M) or most often, both parameters.

TABLE 14 Catalytic activity of FIXa variants (k_(cat)/K_(M)) Mutation(Mature FIX Mutation (Chymotrypsin k_(cat)/K_(M) ±S.D. % of WTNumbering) Numbering) (M⁻¹s⁻¹) (M⁻¹s⁻¹) % CV k_(cat)/K_(M) n BeneFIXBenefix ® BeneFIX Benefix ® 4.1E+07 2.1E+07 51%  91% 125 Coagulation FIX(T148A) Coagulation FIX (T[148]A) Plasma Purified FIXa Plasma PurifiedFIXa 5.2E+07 2.2E+07 41% 117% 120 Catalyst Biosciences WT CatalystBiosciences WT 4.5E+07 2.5E+07 56% 100% 31 N157D N[157]D 2.9E+07 8.1E+0628%  64% 2 Y155F Y[155]F 4.1E+07 1.3E+05  0%  93% 2 A103N/N105S/Y155FA[103]N/N[105]S/Y[155]F 3.9E+07 1.4E+06  4%  88% 2 D104N/K106S/Y155FD[104]N/K[106]S/Y[155]F 3.6E+07 1.0E+06  3%  81% 2 A103N/N105SA[103]N/N[105]S 3.7E+07 1.4E+07 38%  82% 9 D104N/K106S D[104]N/K[106]S3.8E+07 1.3E+07 34%  86% 9 K106N/V108S K[106]N/V[108]S 2.8E+07 6.7E+0624%  62% 7 D85N D[85]N 7.3E+07 2.8E+07 38% 164% 15 T148A T[148]A 4.0E+072.5E+07 62%  89% 30 T148A† T[148]A† 2.3E+07 7.6E+06 33%  52% 7 K5A K[5]A5.6E+07 4.5E+06  8% 125% 2 D64N D[64]N 1.0E+07 1.9E+06 19%  22% 2 D64AD[64]A 2.5E+06 1.1E+06 47%  5% 2 N167D N[167]D 3.1E+07 1.1E+07 34%  69%2 N167Q N[167]Q 3.5E+07 1.9E+07 53%  79% 4 S61A S[61]A 4.8E+07 2.5E+0752% 108% 4 S53A S[53]A 3.5E+07 1.7E+07 48%  78% 3 T159A T[159]A 3.7E+071.2E+07 33%  82% 3 T169A T[169]A 4.7E+07 2.0E+07 43% 106% 3 T172AT[172]A 5.0E+07 2.6E+07 52% 112% 3 T179A T[179]A 5.5E+07 1.3E+07 23%122% 3 Y155H Y[155]H 5.0E+07 1.4E+07 27% 113% 3 Y155Q Y[155]Q 5.4E+072.0E+07 36% 121% 3 S158A S[158]A 3.6E+07 1.1E+06  3%  81% 2 S158DS[158]D 4.0E+07 9.3E+05  2%  89% 2 S158E S[158]E 3.7E+07 3.5E+06  9% 82% 2 N157Q N[157]Q 3.2E+07 2.8E+06  9%  72% 2 D203N/F205T D39N/F41T2.2E+07 1.2E+07 53%  50% 12 D85N/D203N/F205T D[85]N/D39N/F41T 3.0E+076.4E+06 22%  66% 5 K228N K63N 3.6E+07 1.7E+07 49%  80% 13 D85N/K228ND[85]N/K63N 4.6E+07 1.5E+07 32% 104% 6 A103N/N105S/K228NA[103]N/N[105]S/K63N 2.9E+07 1.0E+07 35%  64% 3 D104N/K106S/K228ND[104]N/K[106]S/K63N 2.6E+07 7.6E+06 29%  59% 3 Y155F/K228N Y[155]F/K63N4.5E+07 2.4E+06  5% 101% 2 D104N/K106S/Y155F/ D[104]N/K[106]S/Y[155]F/5.9E+07 1.1E+07 19% 132% 2 K228N K63N 1251S I86S 5.9E+07 1.2E+07 21%132% 13 D85N/I251S D[85]N/I86S 5.6E+07 1.1E+07 20% 124% 5D85N/D104N/K106S/ D[85]N/D[104]N/K[106]S/ 3.3E+07 6.4E+06 19%  75% 5I251S I86S A103N/N105S/I251S A[103]N/N[105]S/I86S 3.9E+07 2.6E+07 67% 87% 3 D104N/K106S/I251S D[104]N/K[106]S/I86S 2.9E+07 1.1E+06  4%  66% 2Y155F/I251S Y[155]F/I86S 6.7E+07 5.9E+06  9% 149% 2 A262S A95bS 2.4E+071.0E+07 42%  54% 8 K413N K243N 2.9E+07 1.7E+07 58%  64% 5 E410N E240N1.3E+08 8.6E+07 65% 297% 21 E410N* E240N* 3.0E+07 1.1E+07 36%  66% 11E239N E74N 2.0E+07 1.1E+07 58%  44% 9 T241N/H243S T76N/H78S 1.9E+075.7E+05  3%  42% 2 K247N/N249S K82N/N84S 5.4E+07 1.7E+07 32% 122% 11Y155F/K247N/N249S Y[155]F/K82N/N84S 5.1E+07 9.6E+06 19% 113% 4A103N/N105S/K247N/ A[103]N/N[105]S/K82N/ 4.0E+07 5.2E+06 13%  90% 6N249S N84S D104N/K106S/K247N/ D[104]N/K[106]S/K82N/ 3.2E+07 3.3E+06 10% 72% 2 N249S N84S D104N/K106S/Y155F/ D[104]N/K[106]S/Y[155]F/ 3.2E+071.1E+07 36%  71% 3 K247N/N249S K82N/N84S L321N L153N 1.6E+07 2.0E+06 13% 35% 2 F314N/H315S F145N/H147S No n.d. n.d.  0% 4 Activity S319N/L321SS151N/L153S 2.8E+07 2.2E+07 78%  64% 3 N260S N95S 1.8E+07 1.2E+07 66% 39% 13 D104N/K106S/N260S D[104]N/K[106]S/N95S 1.3E+07 6.6E+06 51%  29%2 Y155F/N260S Y[155]F/N95S 1.9E+07 1.6E+07 83%  43% 2 D104N/K106S/Y155F/D[104]N/K[106]S/Y[155]F/ 4.3E+06 2.0E+06 46%  10% 2 N260S N95S Y284NY117N 3.5E+07 1.5E+07 42%  78% 8 G317N G149N No n.d. n.d.  0% 5 ActivityR318N/A320S R150N/A152S No n.d. n.d.  0% 8 Activity R318A R150A 4.9E+077.4E+06 15% 108% 3 R318E R150E 1.7E+07 4.2E+06 25%  38% 3 R318Y R150Y7.0E+07 7.0E+06 10% 156% 3 R312Q R143Q 1.1E+07 1.8E+06 17%  23% 3 R312AR143A 4.6E+06 9.3E+05 20%  10% 2 R312Y R143Y 1.2E+07 4.2E+06 36%  27% 2R312L R143L 2.4E+07 9.4E+06 39%  54% 2 V202M V38M 6.6E+07 2.6E+07 39%148% 2 V202Y V38Y 2.5E+07 1.6E+06  6%  56% 2 D203M D39M 4.5E+07 1.9E+0742% 101% 5 D203Y D39Y 3.0E+07 2.8E+06  9%  67% 4 A204M A40M 1.8E+071.2E+07 67%  40% 5 A204Y A40Y 4.6E+07 7.6E+06 16% 103% 2 K400A/R403AK230A/R233A 5.3E+06 6.9E+05 13%  12% 2 K400E/R403E K230E/R233E No n.d.n.d.  0% 4 Activity R403A R233A 1.4E+07 3.0E+06 22%  31% 7 R403E R233E5.5E+06 1.5E+06 28%  12% 6 K400A K230A 2.0E+07 3.1E+06 16%  44% 2 K400EK230E 9.5E+06 1.1E+06 12%  21% 2 K293E K126E 8.1E+06 5.4E+05  7%  18% 2K293A K126A 2.1E+07 4.4E+06 21%  46% 2 R333A R165A No n.d. n.d.  0% 2Activity R333E R165E No n.d. n.d.  0% 2 Activity R338A R170A 1.6E+082.5E+07 15% 361% 2 R338E R170E 1.8E+08 8.3E+07 45% 408% 10 R338A/R403AR170A/R233A 5.3E+07 1.3E+07 24% 119% 6 R338E/R403E R170E/R233E 6.2E+078.8E+06 14% 138% 2 K293A/R403A K126A/R233A 5.7E+06 1.4E+06 25%  13% 2K293E/R403E K126E/R233E 1.3E+06 8.5E+04  6%  3% 2 K293A/R338A/R403AK126A/R170A/R233A 2.5E+07 9.5E+06 39%  55% 2 K293E/R338E/R403EK126E/R170E/R233E 1.7E+07 5.7E+05  3%  37% 2 R318A/R403A R150A/R233A1.5E+07 1.3E+06  9%  33% 2 R318E/R403E R150E/R233E 1.2E+06 3.8E+05 33% 3% 2 R318Y/E410N R150Y/E240N 7.5E+07 2.7E+07 35% 168% 21 R338E/E410NR170E/E240N 4.6E+08 1.7E+08 38% 1018%  8 R338E/R403E/E410NR170E/R233E/E240N 7.8E+07 3.7E+07 47% 175% 7 R318Y/R338E/R403ER150Y/R170E/R233E 6.5E+07 4.6E+06  7% 145% 2 D203N/F205T/K228ND39N/F41T/K63N 1.4E+07 2.5E+06 18%  31% 2 D203N/F205T/E410ND39N/F41T/E240N 4.2E+07 1.7E+07 40%  94% 6 D203N/F205T/R338ED39N/F41T/R170E 1.0E+08 2.3E+07 22% 234% 2 D203N/F205T/R338AD39N/F41T/R170A 6.2E+07 1.4E+07 22% 139% 3 D203N/F205T/R318YD39N/F41T/R150Y 2.0E+07 2.5E+06 12%  45% 4 D203N/F205T/R338E/D39N/F41T/R170E/R233E 1.9E+07 4.8E+06 25%  42% 2 R403E K228N/E410NK63N/E240N 8.5E+07 3.4E+07 40% 190% 10 K228N/R338E K63N/R170E 2.1E+086.1E+07 29% 469% 2 K228N/R338A K63N/R170A 2.1E+08 4.6E+07 22% 473% 2K228N/R318Y K63N/R150Y 4.7E+07 6.5E+06 14% 105% 5 K228N/R338E/R403EK63N/R170E/R233E 4.8E+07 8.6E+06 18% 108% 2 R403E/E410N R233E/E240N2.1E+07 1.7E+06  8%  47% 2 R318Y/R338E/E410N R150Y/R170E/E240N 3.4E+081.4E+08 39% 770% 26 D104N/K106S/R318Y/ D[104]N/K[106]S/R150Y/ 2.6E+085.9E+07 23% 581% 4 R338E/E410N R170E/E240N Y155F/R318Y/R338E/Y[155]F/R150Y/R170E/ 3.7E+08 1.3E+08 33% 835% 5 E410N E240NK228N/R318Y/E410N K63N/R150Y/E240N 1.2E+08 2.6E+07 22% 272% 4R318Y/R403E/E410N R150Y/R233E/E240N 2.7E+07 3.8E+06 14%  59% 3R318Y/R338E/R403E/ R150Y/R170E/R233E/E240N 1.2E+08 8.1E+07 69% 262% 14E410N A103N/N105S/R318Y/ A[103]N/N[105]S/R150Y/ 1.5E+08 7.3E+07 50% 327%5 R338E/R403E/E410N R170E/R233E/E240N D104N/K106S/R318Y/D[104]N/K[106]S/R150Y/ 1.7E+08 7.9E+07 47% 377% 3 R338E/R403E/E410NR170E/R233E/E240N Y155F/R318Y/R338E/ Y[155]F/R150Y/R170E/ 1.9E+085.0E+07 27% 418% 4 R403E/E410N R233E/E240N A103N/N105S/Y155F/A[103]N/N[105]S/Y[155]F/ 1.3E+08 1.8E+06  1% 283% 2 R318Y/R338E/R403E/R150Y/R170E/R233E/E240N E410N D104N/K106S/Y155F/D[104]N/K[106]S/Y[155]F/ 1.8E+08 9.1E+06  5% 394% 2 R318Y/R338E/R403E/R150Y/R170E/R233E/E240N E410N D203N/F205T/R318Y/ D39N/F41T/R150Y/E240N3.9E+07 2.0E+07 52%  88% 6 E410N R333S R165S 1.1E+05 5.5E+04 51%  0.2% 3R338L R170L 2.0E+08 2.3E+07 11% 444% 3 K316N K148N 6.2E+06 4.2E+06 69% 14% 3 K316A K148A 6.1E+06 8.2E+05 13%  14% 3 K316E K148E 7.1E+051.4E+05 19%  2% 3 K316S K148S 3.9E+06 6.2E+05 16%  9% 3 K316M K148M3.1E+07 1.4E+07 46%  70% 3 E239S E74S 3.4E+07 1.8E+07 52%  75% 3 E239AE74A 4.9E+07 6.2E+06 13% 110% 3 E239R E74R 5.6E+07 1.1E+07 19% 126% 3E239K E74K 5.1E+07 5.1E+06 10% 114% 3 H257F H92F 4.8E+07 6.6E+06 14%108% 3 H257Y H92Y 3.4E+07 9.1E+06 27%  75% 3 H257E H92E 2.7E+07 1.5E+0757%  60% 3 H257S H92S 3.5E+07 1.3E+07 36%  78% 3 T412A T242A 4.6E+072.8E+07 62% 103% 5 T412V T242V 5.8E+07 3.2E+07 55% 130% 8 E410N/T412AE240N/T242A 8.0E+07 1.7E+07 21% 178% 4 E410N/T412V E240N/T242V 8.8E+072.7E+07 30% 197% 4 E410Q E240Q 1.2E+08 7.6E+07 63% 269% 4 E410S E240S1.1E+08 6.6E+07 60% 246% 12 E410A E240A 1.1E+08 5.6E+07 50% 248% 10E410D E240D 6.0E+07 1.6E+07 27% 134% 4 N346D N178D 1.9E+07 8.5E+06 44% 43% 4 Y155F/N346D Y[155]F/N178D 1.3E+07 6.8E+06 53%  29% 2 N346Y N178Y9.8E+07 2.3E+07 24% 218% 8 Y345A Y177A 1.5E+07 6.3E+06 43%  32% 4 Y345TY177T 5.0E+07 2.5E+07 50% 112% 4 T343R T175R 1.7E+08 1.1E+08 66% 372% 9T343E T175E 4.0E+07 2.3E+07 58%  88% 4 T343Q T175Q 7.1E+07 2.2E+07 30%159% 3 F342I F174I 5.4E+07 2.9E+07 54% 121% 3 T343R/Y345T T175R/Y177T9.3E+07 1.8E+07 19% 208% 3 R318Y/R338E R150Y/R170E 1.5E+08 5.3E+07 36%331% 4 Y259F/K265T/Y345T Y94F/K98T/Y177T 5.6E+07 1.2E+07 21% 126% 2K228N/I251S K63N/I86S 2.2E+07 5.7E+05  3%  50% 2 K228N/R318Y/R338E/K63N/R150Y/R170E/R233E/ 1.6E+08 6.1E+07 39% 349% 3 R403E/E410N E240NY155F/K228N/R318Y/ Y[155]F/K63N/R150Y/R170E/ 2.0E+08 9.3E+06  5% 453% 2R338E/R403E/E410N R233E/E240N D85N/K228N/R318Y/ D[85]N/K63N/R150Y/R170E/1.6E+08 2.3E+07 15% 346% 2 R338E/R403E/E410N R233E/E240NI251S/R318Y/R338E/ I86S/R150Y/R170E/R233E/ 1.5E+08 4.2E+07 27% 344% 4R403E/E410N E240N D104N/K106S/I251S/ D[104]N/K[106]S/I86S/R150Y/ 1.2E+082.0E+07 16% 271% 8 R318Y/R338E/R403E/ R170E/R233E/E240N E410NY155F/I251S/R318Y/ Y[155]F/I86S/R150Y/R170E/ 1.7E+08 9.2E+06  6% 374% 2R338E/R403E/E410N R233E/E240N I251S/R318Y/R338E/ I86S/R150Y/R170E/E240N3.8E+08 6.1E+07 16% 851% 7 E410N D104N/K106S/I251S/D[104]N/K[106]S/I86S/R150Y/ 1.3E+08 3.2E+07 24% 300% 3 R318Y/R338E/E410NR170E/E240N F314N/K316S F145N/K148S 8.8E+04 8.2E+04 94%  0.2% 2K247N/N249S/R318Y/ K82N/N84S/R150Y/R170E/ 1.5E+08 4.7E+07 30% 341% 6R338E/R403E/E410N R233E/E240N Y155F/K247N/N249S/Y[155]F/K82N/N84S/R150Y/ 1.8E+08 6.1E+07 33% 408% 6 R318Y/R338E/R403E/R170E/R233E/E240N E410N A103N/N105S/K247N/ A[103]N/N[105]S/K82N/ 1.0E+087.6E+06  7% 232% 2 N249S/R318Y/R338E/ N84S/R150Y/R170E/R233E/R403E/E410N E240N D104N/K106S/K247N/ D[104]N/K[106]S/K82N/ 8.8E+076.5E+06  7% 197% 2 N249S/R318Y/R338E/ N84S/R150Y/R170E/R233E/R403E/E410N E240N K247N/N249S/R318Y/ K82N/N84S/R150Y/R170E/ 2.3E+086.6E+07 28% 516% 6 R338E/E410N E240N Y155F/K247N/N249S/Y[155]F/K82N/N84S/R150Y/ 3.0E+08 1.3E+08 42% 674% 7 R318Y/R338E/E410NR170E/E240N R318Y/R338E/R403E/ R150Y/R170E/R233E/E240S 1.8E+08 6.2E+0734% 401% 4 E410S R318Y/R338E/E410S R150Y/R170E/E240S 3.3E+08 1.2E+08 37%730% 8 K228N/K247N/N249S K63N/K82N/N84S 3.8E+07 1.2E+07 32%  86% 2D104N/K106S/Y155F/ D[104]N/K[106]S/Y[155]F/ 6.3E+07 3.3E+06  5% 142% 2K228N/K247N/N249S K63N/K82N/N84S D104N/K106S/K228N/D[104]N/K[106]S/K63N/ 2.3E+07 1.1E+07 48%  51% 5 K247N/N249S K82N/N84SY155F/K228N/K247N/ Y[155]F/K63N/K82N/N84S 5.3E+07 5.5E+06 10% 118% 2N249S K228N/K247N/N249S/ K63N/K82N/N84S/R150Y/ 1.2E+08 3.8E+07 33% 258%3 R318Y/R338E/R403E/ R170E/R233E/E240N E410N R318Y/R338E/R403E/R150Y/R170E/R233E/E240N/ 1.9E+08 5.0E+07 26% 424% 4 E410N/T412V T242VR318Y/R338E/R403E/ R150Y/R170E/R233E/E240N/ 2.6E+08 7.4E+07 29% 577% 4E410N/T412A T242A R318Y/R338E/R403E/ R150Y/R170E/R233E/T242A 8.0E+073.4E+07 42% 178% 4 T412A R318Y/R338E/T412A R150Y/R170E/T242A 3.0E+088.3E+07 28% 661% 6 R318Y/R338E/E410N/ R150Y/R170E/E240N/T242V 2.4E+081.4E+08 60% 536% 4 T412V N260S/R318Y/R338E/ N95S/R150Y/R170E/R233E/5.3E+07 6.6E+05  1% 117% 2 R403E/E410N E240N D104N/K106S/N260S/D[104]N/K[106]S/N95S/ 8.8E+07 7.9E+06  9% 196% 2 R318Y/R338E/R403E/R150Y/R170E/R233E/E240N E410N Y155F/N260S/R318Y/Y[155]F/N95S/R150Y/R170E/ 7.0E+07 2.4E+07 35% 156% 2 R338E/R403E/E410NR233E/E240N R318Y/R338E/N346D/ R150Y/R170E/N178D/R233E/ 3.1E+07 9.1E+0630%  68% 2 R403E/E410N E240N Y155F/R318Y/R338E/ Y[155]F/R150Y/R170E/6.2E+07 1.8E+07 30% 139% 2 N346D/R403E/E410N N178D/R233E/E240NK247N/N249S/N260S K82N/N84S/N95S 2.9E+07 2.6E+06  9%  64% 2Y155F/K247N/N249S/ Y[155]F/K82N/N84S/N95S 1.9E+07 4.2E+06 22%  43% 2N260S D104N/K106S/K247N/ D[104]N/K[106]S/K82N/ 9.8E+06 3.0E+06 30%  22%2 N249S/N260S N84S/N95S D104N/K106S/Y155F/ D[104]N/K[106]S/Y[155]F/8.2E+06 3.9E+06 47%  18% 2 K247N/N249S/N260S K82N/N84S/N95SK247N/N249S/N260S/ K82N/N84S/N95S/R150Y/ 9.7E+07 8.7E+06  9% 217% 2R318Y/R338E/R403E/ R170E/R233E/E240N E410N Y155F/N260S/N346DY[155]F/N95S/N178D 2.2E+06 7.4E+05 34%  5% 2 R318Y/R338E/T343R/R150Y/R170E/T175R/R233E/ 5.4E+08 1.6E+08 29% 1217%  3 R403E/E410N E240NR338E/T343R R170E/T175R 6.0E+08 1.7E+08 29% 1329%  4 †produced in BHK-21cells; *80% glycosylated form of E410N

TABLE 15 Catalytic activity of FIXa variants (k_(cat)/K_(M)) % ofMutation (Mature FIX Mutation (Chymotrypsin k_(cat)/K_(M) ±S.D. WTNumbering) Numbering) (M⁻¹s⁻¹) (M⁻¹s⁻¹) % CV k_(cat)/K_(M) n BeneFIXBenefix ® BeneFIX Benefix ® 4.3E+07 2.3E+07 54% 92% 140 Coagulation FIX(T148A) Coagulation FIX (T[148]A) Plasma Purified FIXa Plasma PurifiedFIXa 5.6E+07 2.6E+07 46% 122% 200 Catalyst Biosciences WT CatalystBiosciences WT 4.6E+07 2.5E+07 54% 100% 33 N157D N[157]D 2.9E+07 8.1E+0628% 62% 2 Y155F Y[155]F 4.1E+07 1.3E+05 0% 90% 2 A103N/N105S/Y155FA[103]N/N[105]S/Y[155]F 3.9E+07 1.4E+06 4% 85% 2 D104N/K106S/Y155FD[104]N/K[106]S/Y[155]F 3.6E+07 1.0E+06 3% 78% 2 A103N/N105SA[103]N/N[105]S 3.7E+07 1.4E+07 38% 80% 9 D104N/K106S D[104]N/K[106]S3.8E+07 1.3E+07 34% 83% 9 K106N/V108S K[106]N/V[108]S 2.8E+07 6.7E+0624% 60% 7 D85N D[85]N 7.0E+07 2.7E+07 39% 153% 17 T148A T[148]A 4.0E+072.2E+07 54% 88% 44 T148A† T[148]A† 2.3E+07 7.6E+06 33% 50% 7 K5A K[5]A5.5E+07 9.3E+06 17% 120% 4 D64N D[64]N 1.0E+07 1.9E+06 19% 22% 2 D64AD[64]A 2.5E+06 1.1E+06 47% 5% 2 N167D N[167]D 3.1E+07 1.1E+07 34% 67% 2N167Q N[167]Q 3.5E+07 1.9E+07 53% 76% 4 S61A S[61]A 4.8E+07 2.5E+07 52%105% 4 S53A S[53]A 3.5E+07 1.7E+07 48% 76% 3 T159A T[159]A 3.7E+071.2E+07 33% 80% 3 T169A T[169]A 4.7E+07 2.0E+07 43% 103% 3 T172A T[172]A5.0E+07 2.6E+07 52% 109% 3 T179A T[179]A 5.5E+07 1.3E+07 23% 119% 3Y155H Y[155]H 5.0E+07 1.4E+07 27% 109% 3 Y155Q Y[155]Q 5.4E+07 2.0E+0736% 117% 3 S158A S[158]A 3.6E+07 1.1E+06 3% 79% 2 S158D S[158]D 4.0E+079.3E+05 2% 86% 2 S158E S[158]E 3.7E+07 3.5E+06 9% 80% 2 N157Q N[157]Q3.2E+07 2.8E+06 9% 70% 2 D203N/F205T D39N/F41T 2.2E+07 1.2E+07 53% 49%12 D85N/D203N/F205T D[85]N/D39N/F41T 3.0E+07 6.4E+06 22% 64% 5 K228NK63N 3.6E+07 1.7E+07 49% 77% 13 D85N/K228N D[85]N/K63N 4.6E+07 1.5E+0732% 101% 6 A103N/N105S/K228N A[103]N/N[105]S/K63N 2.9E+07 1.0E+07 35%63% 3 D104N/K106S/K228N D[104]N/K[106]S/K63N 2.6E+07 7.6E+06 29% 57% 3Y155F/K228N Y[155]F/K63N 4.5E+07 2.4E+06 5% 98% 2D104N/K106S/Y155F/K228N D[104]N/K[106]S/Y[155]F/K63N 5.9E+07 1.1E+07 19%129% 2 I251S I86S 5.9E+07 1.2E+07 21% 128% 13 D85N/I251S D[85]N/I86S5.6E+07 1.1E+07 20% 121% 5 D85N/D104N/K106S/I251SD[85]N/D[104]N/K[106]S/ 3.3E+07 6.4E+06 19% 73% 5 I86S A103N/N105S/I251SA[103]N/N[105]S/I86S 3.9E+07 2.6E+07 67% 84% 3 D104N/K106S/I251SD[104]N/K[106]S/I86S 2.9E+07 1.1E+06 4% 64% 2 Y155F/I251S Y[155]F/I86S6.7E+07 5.9E+06 9% 145% 2 A262S A95bS 2.4E+07 1.0E+07 42% 52% 8 K413NK243N 2.8E+07 1.4E+07 51% 60% 7 E410N E240N 1.3E+08 7.7E+07 60% 277% 27E410N* E240N* 3.0E+07 1.1E+07 36% 65% 10 E239N E74N 2.0E+07 1.1E+07 58%43% 9 T241N/H243S T76N/H78S 1.9E+07 5.7E+05 3% 41% 2 K247N/N249SK82N/N84S 5.4E+07 1.7E+07 32% 118% 11 Y155F/K247N/N249SY[155]F/K82N/N84S 5.1E+07 9.6E+06 19% 110% 4 A103N/N105S/K247N/A[103]N/N[105]S/K82N/ 4.0E+07 5.2E+06 13% 87% 6 N249S N84SD104N/K106S/K247N/ D[104]N/K[106]S/K82N/ 3.2E+07 3.3E+06 10% 69% 2 N249SN84S D104N/K106S/Y155F/ D[104]N/K[106]S/Y[155]F/ 3.2E+07 1.1E+07 36% 69%3 K247N/N249S K82N/N84S L321N L153N 1.6E+07 2.0E+06 13% 34% 2F314N/H315S F145N/H147S 4.4E+05 3.7E+04 8% 1% 2 K392N/K394S K222N/K224S0.0E+00 n.d. n.d. 0% 0 S319N/L321S S151N/L153S 2.8E+07 2.2E+07 78% 62% 3N260S N95S 1.8E+07 1.2E+07 66% 38% 13 D104N/K106S/N260SD[104]N/K[106]S/N95S 1.3E+07 6.6E+06 51% 28% 2 Y155F/N260S Y[155]F/N95S1.9E+07 1.6E+07 83% 42% 2 D104N/K106S/Y155F/ D[104]N/K[106]S/Y[155]F/4.3E+06 2.0E+06 46% 9% 2 N260S N95S Y284N Y117N 3.5E+07 1.5E+07 42% 76%8 G317N G149N 4.6E+04 n.d. n.d. 0% 1 R318N/A320S R150N/A152S 2.3E+052.1E+05 89% 1% 3 R318A R150A 4.5E+07 6.4E+06 14% 98% 2 R318E R150E1.7E+07 4.2E+06 25% 37% 3 R318Y R150Y 7.0E+07 7.0E+06 10% 151% 3 R312QR143Q 1.1E+07 1.8E+06 17% 23% 3 R312A R143A 4.6E+06 9.3E+05 20% 10% 2R312Y R143Y 1.2E+07 4.2E+06 36% 26% 2 R312L R143L 2.4E+07 9.4E+06 39%53% 2 V202M V38M 6.6E+07 2.6E+07 39% 143% 2 V202Y V38Y 2.5E+07 1.6E+066% 55% 2 D203M D39M 4.5E+07 1.9E+07 42% 98% 5 D203Y D39Y 3.0E+07 2.8E+069% 65% 4 A204M A40M 1.8E+07 1.2E+07 67% 39% 5 A204Y A40Y 4.6E+07 7.6E+0616% 100% 2 K400A/R403A K230A/R233A 5.3E+06 6.9E+05 13% 12% 2 K400E/R403EK230E/R233E 4.3E+05 3.1E+04 7% 1% 3 R403A R233A 1.4E+07 3.0E+06 22% 30%7 R403E R233E 5.5E+06 1.5E+06 28% 12% 6 K400A K230A 2.0E+07 3.1E+06 16%43% 2 K400E K230E 9.5E+06 1.1E+06 12% 21% 2 K293E K126E 8.1E+06 5.4E+057% 17% 2 K293A K126A 2.1E+07 4.4E+06 21% 45% 2 R333A R165A 1.6E+051.1E+04 7% 0% 2 R333E R165E 1.3E+04 n.d. n.d. 0% 1 R338A R170A 1.6E+082.5E+07 15% 350% 2 R338E R170E 1.8E+08 8.3E+07 45% 396% 10 R338A/R403AR170A/R233A 5.3E+07 1.3E+07 24% 115% 6 R338E/R403E R170E/R233E 6.2E+078.8E+06 14% 134% 2 K293A/R403A K126A/R233A 5.7E+06 1.4E+06 25% 12% 2K293E/R403E K126E/R233E 1.3E+06 8.5E+04 6% 3% 2 K293A/R338A/R403AK126A/R170A/R233A 2.5E+07 9.5E+06 39% 53% 2 K293E/R338E/R403EK126E/R170E/R233E 1.7E+07 5.7E+05 3% 36% 2 R318A/R403A R150A/R233A1.5E+07 1.3E+06 9% 32% 2 R318E/R403E R150E/R233E 1.2E+06 3.8E+05 33% 3%2 R318Y/E410N R150Y/E240N 7.5E+07 2.7E+07 35% 163% 21 R338E/E410NR170E/E240N 4.4E+08 1.5E+08 33% 950% 12 R338E/R403E/E410NR170E/R233E/E240N 1.9E+08 1.4E+08 72% 411% 17 Y155F/R338E/R403E/Y[155]F/R170E/R233E/ 1.8E+08 6.0E+07 32% 401% 2 E410N E240NR318Y/R338E/R403E R150Y/R170E/R233E 6.2E+07 6.3E+06 10% 134% 3Y155F/R318Y/R338E/ Y[155]F/R150Y/R170E/ 8.7E+07 5.1E+07 58% 189% 2 R403ER233E D203N/F205T/K228N D39N/F41T/K63N 1.4E+07 2.5E+06 18% 30% 2D203N/F205T/E410N D39N/F41T/E240N 4.2E+07 1.7E+07 40% 91% 6D203N/F205T/R338E D39N/F41T/R170E 1.0E+08 2.3E+07 22% 228% 2D203N/F205T/R338A D39N/F41T/R170A 6.2E+07 1.4E+07 22% 135% 3D203N/F205T/R318Y D39N/F41T/R150Y 2.0E+07 2.5E+06 12% 44% 4D203N/F205T/R338E/ D39N/F41T/R170E/R233E 1.9E+07 4.8E+06 25% 41% 2 R403EK228N/E410N K63N/E240N 8.5E+07 3.4E+07 40% 184% 10 K228N/R338EK63N/R170E 2.1E+08 6.1E+07 29% 455% 2 K228N/R338A K63N/R170A 2.1E+084.6E+07 22% 459% 2 K228N/R318Y K63N/R150Y 4.7E+07 6.5E+06 14% 102% 5K228N/R338E/R403E K63N/R170E/R233E 4.8E+07 8.6E+06 18% 105% 2R403E/E410N R233E/E240N 2.1E+07 1.7E+06 8% 46% 2 R318Y/R338E/E410NR150Y/R170E/E240N 3.4E+08 1.2E+08 37% 727% 42 D104N/K106S/R318Y/D[104]N/K[106]S/R150Y/ 2.6E+08 5.9E+07 23% 564% 4 R338E/E410NR170E/E240N Y155F/R318Y/R338E/ Y[155]F/R150Y/R170E/ 3.7E+08 1.3E+08 33%810% 5 E410N E240N K228N/R318Y/E410N K63N/R150Y/E240N 1.2E+08 2.6E+0722% 264% 4 R318Y/R403E/E410N R150Y/R233E/E240N 2.5E+07 4.7E+06 19% 54% 5Y155F/R318Y/R403E/ Y[155]F/R150Y/R233E/ 3.6E+07 2.9E+07 82% 78% 2 E410NE240N R318Y/R338E/R403E/ R150Y/R170E/R233E/E240N 1.5E+08 8.2E+07 56%320% 26 E410N A103N/N105S/R318Y/ A[103]N/N[105]S/R150Y/ 1.5E+08 7.3E+0750% 318% 5 R338E/R403E/E410N R170E/R233E/E240N D104N/K106S/R318Y/D[104]N/K[106]S/R150Y/ 1.7E+08 7.9E+07 47% 366% 3 R338E/R403E/E410NR170E/R233E/E240N Y155F/R318Y/R338E/ Y[155]F/R150Y/R170E/ 1.9E+085.0E+07 27% 406% 4 R403E/E410N R233E/E240N A103N/N105S/Y155F/R318Y/A[103]N/N[105]S/Y[155]F/ 1.3E+08 1.8E+06 1% 274% 2 R338E/R403E/E410NR150Y/R170E/R233E/E240N D104N/K106S/Y155F/R318Y/D[104]N/K[106]S/Y[155]F/ 1.8E+08 9.1E+06 5% 382% 2 R338E/R403E/E410NR150Y/R170E/R233E/E240N D203N/F205T/R318Y/ D39N/F41T/R150Y/E240N 3.9E+072.0E+07 52% 85% 6 E410N R333S R165S 1.1E+05 5.5E+04 51% 0% 3 R338L R170L2.0E+08 2.3E+07 11% 431% 3 K316N K148N 6.2E+06 4.2E+06 69% 13% 3 K316AK148A 6.1E+06 8.2E+05 13% 13% 3 K316E K148E 7.1E+05 1.4E+05 19% 2% 3K316S K148S 3.9E+06 6.2E+05 16% 9% 3 K316M K148M 3.1E+07 1.4E+07 46% 68%3 E239S E74S 3.4E+07 1.8E+07 52% 73% 3 E239A E74A 4.9E+07 6.2E+06 13%107% 3 E239R E74R 5.6E+07 1.1E+07 19% 122% 3 E239K E74K 5.1E+07 5.1E+0610% 111% 3 H257F H92F 4.8E+07 6.6E+06 14% 105% 3 H257Y H92Y 3.4E+079.1E+06 27% 73% 3 H257E H92E 2.7E+07 1.5E+07 57% 58% 3 H257S H92S3.5E+07 1.3E+07 36% 76% 3 T412A T242A 4.6E+07 2.8E+07 62% 100% 5 T412VT242V 5.8E+07 3.2E+07 55% 126% 8 E410N/T412A E240N/T242A 8.0E+07 1.7E+0721% 173% 4 E410N/T412V E240N/T242V 8.8E+07 2.7E+07 30% 192% 4 E410QE240Q 1.2E+08 7.6E+07 63% 261% 4 E410S E240S 1.1E+08 6.6E+07 60% 239% 12E410A E240A 1.1E+08 5.6E+07 50% 241% 10 E410D E240D 6.0E+07 1.6E+07 27%130% 4 N346D N178D 1.9E+07 8.5E+06 44% 42% 4 Y155F/N346D Y[155]F/N178D1.3E+07 6.8E+06 53% 28% 2 N346Y N178Y 9.8E+07 2.3E+07 24% 212% 8 Y345AY177A 1.5E+07 6.3E+06 43% 32% 4 Y345T Y177T 5.0E+07 2.5E+07 50% 108% 4T343R T175R 1.4E+08 1.0E+08 70% 313% 12 T343E T175E 4.0E+07 2.3E+07 58%86% 4 T343Q T175Q 7.1E+07 2.2E+07 30% 154% 3 F342I F174I 5.4E+07 2.9E+0754% 118% 3 T343R/Y345T T175R/Y177T 9.3E+07 1.8E+07 19% 202% 3R318Y/R338E R150Y/R170E 1.5E+08 5.3E+07 36% 322% 4 Y259F/K265T/Y345TY94F/K98T/Y177T 5.6E+07 1.2E+07 21% 122% 2 K228N/I251S K63N/I86S 2.2E+075.7E+05 3% 48% 2 K228N/R318Y/R338E/ K63N/R150Y/R170E/R233E/ 1.6E+086.1E+07 39% 339% 3 R403E/E410N E240N Y155F/K228N/R318Y/Y[155]F/K63N/R150Y/R170E/ 1.6E+08 4.1E+07 25% 356% 5 R338E/R403E/E410NR233E/E240N D85N/K228N/R318Y/ D[85]N/K63N/R150Y/R170E/ 1.6E+08 2.3E+0715% 336% 2 R338E/R403E/E410N R233E/E240N I251S/R318Y/R338E/I86S/R150Y/R170E/R233E/ 1.5E+08 4.2E+07 27% 334% 4 R403E/E410N E240ND104N/K106S/I251S/R318Y/ D[104]N/K[106]S/I86S/ 1.2E+08 2.0E+07 16% 263%8 R338E/R403E/E410N R150Y/R170E/R233E/E240N Y155F/I251S/R318Y/D[104]N/K[106]S/I86S/ 1.7E+08 9.2E+06 6% 363% 2 R338E/R403E/E410NR150Y/R170E/R233E/E240N I251S/R318Y/R338E/ I86S/R150Y/R170E/E240N3.9E+08 7.4E+07 19% 849% 10 E410N D104N/K106S/I251S/D[104]N/K[106]S/I86S/ 1.3E+08 3.2E+07 24% 291% 3 R318Y/R338E/E410NR150Y/R170E/E240N F314N/K316S F145N/K148S 8.8E+04 8.2E+04 94% 0% 2K247N/N249S/R318Y/ K82N/N84S/R150Y/R170E/ 1.5E+08 4.7E+07 30% 331% 6R338E/R403E/E410N R233E/E240N Y155F/K247N/N249S/R318Y/Y[155]F/K82N/N84S/R150Y/ 1.9E+08 5.7E+07 30% 405% 10 R338E/R403E/E410NR170E/R233E/E240N A103N/N105S/K247N/ A[103]N/N[105]S/K82N/ 1.5E+084.2E+07 28% 324% 6 N249S/R318Y/R338E/ N84S/R150Y/R170E/R233E/R403E/E410N E240N D104N/K106S/K247N/ D[104]N/K[106]S/K82N/ 8.8E+076.5E+06 7% 192% 2 N249S/R318Y/R338E/ N84S/R150Y/R170E/R233E/ R403E/E410NE240N D104N/K106S/Y155F/ D[104]N/K[106]S/Y[155]F/ 1.3E+08 7.3E+07 54%292% 6 K247N/N249S/R318Y/ K82N/N84S/R150Y/R170E/ R338E/R403E/E410NR233E/E240N K247N/N249S/R318Y/ K82N/N84S/R150Y/R170E/E240N 2.3E+086.6E+07 28% 501% 6 R338E/E410N Y155F/K247N/N249S/Y[155]F/K82N/N84S/R150Y/ 3.3E+08 1.3E+08 39% 717% 9 R318Y/R338E/E410NR170E/E240N R318Y/R338E/R403E/ R150Y/R170E/R233E/E240S 2.1E+08 6.1E+0729% 458% 7 E410S R318Y/R338E/E410S R150Y/R170E/E240S 3.3E+08 1.2E+08 37%708% 8 K228N/K247N/N249S K63N/K82N/N84S 3.8E+07 1.2E+07 32% 83% 2D104N/K106S/Y155F/ D[104]N/K[106]S/Y[155]F/ 6.3E+07 3.3E+06 5% 137% 2K228N/K247N/N249S K63N/K82N/N84S D104N/K106S/K228N/D[104]N/K[106]S/K63N/ 2.3E+07 1.1E+07 48% 49% 5 K247N/N249S K82N/N84SY155F/K228N/K247N/ Y[155]F/K63N/K82N/N84S 5.3E+07 5.5E+06 10% 115% 2N249S K228N/K247N/N249S/R318Y/ K63N/K82N/N84S/R150Y/ 1.6E+08 8.4E+07 51%352% 17 R338E/R403E/E410N R170E/R233E/E240N D104N/K106S/K228N/D[104]N/K[106]S/K63N/ 1.1E+08 4.4E+07 40% 239% 7 K247N/N249S/R318Y/K82N/N84S/R150Y/R170E/ R338E/R403E/E410N R233E/E240N Y155F/K228N/K247N/Y[155]F/K63N/K82N/N84S/ 1.2E+08 5.3E+07 44% 263% 5 N249S/R318Y/R338E/R150Y/R170E/R233E/E240N R403E/E410N R318Y/R338E/R403E/R150Y/R170E/R233E/E240N/ 1.6E+08 6.3E+07 40% 342% 6 E410N/T412V T242VR318Y/R338E/R403E/ R150Y/R170E/R233E/E240N/ 2.5E+08 9.2E+07 37% 538% 6E410N/T412A T242A R318Y/R338E/R403E/ R150Y/R170E/R233E/T242A 8.0E+073.4E+07 42% 173% 4 T412A R318Y/R338E/T412A R150Y/R170E/T242A 3.0E+088.3E+07 28% 642% 6 R318Y/R338E/E410N/ R150Y/R170E/E240N/T242V 2.6E+081.2E+08 46% 571% 11 T412V N260S/R318Y/R338E/ N95S/R150Y/R170E/R233E/5.3E+07 6.6E+05 1% 114% 2 R403E/E410N E240N D104N/K106S/N260S/R318Y/D[104]N/K[106]S/N95S/R150Y/ 8.8E+07 7.9E+06 9% 190% 2 R338E/R403E/E410NR170E/R233E/E240N Y155F/N260S/R318Y/ Y[155]F/N95S/R150Y/R170E/ 7.0E+072.4E+07 35% 152% 2 R338E/R403E/E410N R233E/E240N R318Y/R338E/N346D/R150Y/R170E/N178D/R233E/ 3.1E+07 9.1E+06 30% 66% 2 R403E/E410N E240NY155F/R318Y/R338E/ Y[155]F/R150Y/R170E/ 6.2E+07 1.8E+07 30% 135% 2N346D/R403E/E410N N178D/R233E/E240N K247N/N249S/N260S K82N/N84S/N95S2.9E+07 2.6E+06 9% 62% 2 Y155F/K247N/N249S/ Y[155]F/K82N/N84S/N95S1.9E+07 4.2E+06 22% 42% 2 N260S D104N/K106S/K247N/ D[104]N/K[106]S/K82N/9.8E+06 3.0E+06 30% 21% 2 N249S/N260S N84S/N95S D104N/K106S/Y155F/D[104]N/K[106]S/Y[155]F/ 8.2E+06 3.9E+06 47% 18% 2 K247N/N249S/N260SK82N/N84S/N95S K247N/N249S/N260S/R318Y/ K82N/N84S/N95S/R150Y/ 6.7E+072.6E+07 38% 145% 6 R338E/R403E/E410N R170E/R233E/E240NY155F/K247N/N249S/ Y[155]F/K82N/N84S/N95S/ 5.7E+07 3.6E+07 64% 124% 5N260S/R318Y/R338E/ R150Y/R170E/R233E/E240N R403E/E410N Y155F/N260S/N346DY[155]F/N95S/N178D 2.2E+06 7.4E+05 34% 5% 2 R318Y/R338E/T343R/R150Y/R170E/T175R/R233E/ 4.2E+08 1.4E+08 33% 907% 13 R403E/E410N E240NY155F/R318Y/R338E/ Y[155]F/R150Y/R170E/ 3.0E+08 8.3E+07 28% 640% 4T343R/R403E/E410N T175R/R233E/E240N D104N/K106S/R318Y/R338E/D[104]N/K[106]S/R150Y/ 2.2E+08 1.2E+08 52% 487% 5 T343R/R403E/E410NR170E/T175R/R233E/E240N R338E/T343R R170E/T175R 5.2E+08 1.6E+08 31%1120% 7 T343R/N346Y T175R/N178Y 9.6E+07 4.4E+07 46% 208% 11R318Y/R338E/N346Y/ R150Y/R170E/N178Y/R233E/ 1.2E+08 2.1E+07 16% 270% 3R403E/E410N E240N R318Y/R338E/T343R/ R150Y/R170E/T175R/N178Y/ 3.1E+081.1E+08 37% 663% 5 N346Y/R403E/E410N R233E/E240N T343R/N346D T175R/N178D1.6E+07 2.6E+06 16% 36% 2 R318Y/R338E/T343R/ R150Y/R170E/T175R/N178D/8.2E+07 3.2E+06 4% 177% 2 N346D/R403E/E410N R233E/E240NR318Y/R338E/Y345A/ R150Y/R170E/Y177A/R233E/ 8.3E+07 3.6E+07 44% 180% 6R403E/E410N E240N R318Y/R338E/Y345A/ R150Y/R170E/Y177A/N178D/ 2.3E+077.6E+06 33% 49% 3 N346D/R403E/E410N R233E/E240N Y155F/K247N/N249S/Y[155]F/K82N/N84S/R150Y/ 9.5E+07 6.6E+07 69% 206% 5 R318Y/R338E/R403ER170E/R233E K247N/N249S/R318Y/ K82N/N84S/R150Y/R170E/ 2.3E+08 1.6E+0871% 496% 2 R338E/R403E R233E Y155F/K247N/N249S/ Y[155]F/K82N/N84S/R150Y/1.0E+07 4.5E+06 45% 22% 3 R318Y/R403E/E410N R233E/E240NK247N/N249S/R318Y/ K82N/N84S/R150Y/R233E/ 2.7E+07 1.2E+07 44% 58% 10R403E/E410N E240N Y155F/K247N/N249S/ Y[155]F/K82N/N84S/R170E/ 1.1E+082.4E+07 23% 229% 3 R338E/R403E/E410N R233E/E240N K247N/N249S/R338E/K82N/N84S/R170E/R233E/ 1.9E+08 2.9E+07 15% 422% 2 R403E/E410N E240NR318Y/R338E/T343R/ R150Y/R170E/T175R/R233E 1.6E+08 7.4E+07 45% 357% 4R403E Y155F/R318Y/R338E/ Y[155]F/R150Y/R170E/ 2.6E+08 1.7E+08 65% 563% 4T343R/R403E T175R/R233E R318Y/R338E/T343R/ R150Y/R170E/T175R/E240N3.4E+08 1.6E+08 48% 728% 16 E410N Y155F/R318Y/R338E/Y[155]F/R150Y/R170E/ 3.7E+08 1.2E+08 32% 794% 4 T343R/E410N T175R/E240NR318Y/T343R/R403E/ R150Y/T175R/R233E/E240N 5.8E+07 1.8E+07 31% 125% 3E410N Y155F/R318Y/T343R/ Y[155]F/R150Y/T175R/ 2.6E+08 5.0E+07 19% 571% 2R403E/E410N R233E/E240N R338E/T343R/R403E/ R170E/T175R/R233E/E240N3.0E+08 8.2E+07 27% 650% 2 E410N Y155F/R338E/T343R/ Y[155]F/R170E/T175R/2.4E+08 1.0E+08 42% 524% 4 R403E/E410N R233E/E240NY155F/K247N/N249S/R318Y/ Y[155]F/K82N/N84S/R150Y/ 4.0E+08 1.5E+08 37%864% 11 R338E/T343R/R403E/E410N R170E/T175R/R233E/E240NK247N/N249S/R318Y/R338E/ K82N/N84S/R150Y/R170E/ 3.8E+08 1.5E+08 40% 824%5 T343R/R403E/E410N T175R/R233E/E240N K228N/I251S/R318Y/K63N/I86S/R150Y/R170E/ 2.1E+08 7.2E+07 34% 463% 7 R338E/R403E/E410NR233E/E240N Y155F/K228N/I251S/R318Y/ Y[155]F/K63N/I86S/R150Y/ 1.4E+085.0E+07 37% 296% 5 R338E/R403E/E410N R170E/R233E/E240NN260S/R318Y/R338E/ N95S/R150Y/R170E/T175R/ 2.9E+08 1.1E+08 38% 638% 7T343R/R403E/E410N R233E/E240N Y155F/N260S/R318Y/R338E/Y[155]F/N95S/R150Y/R170E/ 1.5E+08 6.0E+07 39% 335% 5 T343R/R403E/E410NT175R/R233E/E240N K228N/K247N/N249S/ K63N/K82N/N84S/R150Y/ 4.1E+081.4E+08 34% 880% 12 R318Y/R338E/T343R/ R170E/T175R/R233E/E240NR403E/E410N Y155F/K228N/K247N/ Y[155]F/K63N/K82N/N84S/ 3.0E+08 1.1E+0837% 646% 5 N249S/R318Y/R338E/ R150Y/R170E/T175R/R233E/ T343R/R403E/E410NE240N Y155F/R338E/T343R/ Y[155]F/R170E/T175R/ 2.0E+08 7.7E+07 39% 429% 5R403E R233E R338E/T343R/R403E R170E/T175R/R233E 3.1E+08 9.6E+07 31% 663%2 Y155F/R338E/T343R/ Y[155]F/R170E/T175R/ 2.9E+08 1.0E+08 35% 629% 6R403E/E410S R233E/E240S Y155F/N260S/R338E/ Y[155]F/N95S/R170E/T175R/9.4E+07 3.1E+07 33% 203% 6 T343R/R403E R233E Y155F/I251S/R338E/Y[155]F/I86S/R170E/T175R/ 3.0E+08 1.6E+07 5% 651% 2 T343R/R403E R233ER318Y/R338E/T343R/ R150Y/R170E/T175R/R233E/ 4.4E+08 1.7E+08 39% 962% 14R403E/E410S E240S Y155F/K247N/N249S/ Y[155]F/K82N/N84S/T175R/ 8.5E+072.7E+07 31% 184% 4 T343R/R403E R233E Y155F/K247N/N249S/R318Y/Y[155]F/K82N/N84S/R150Y/ 2.9E+08 5.0E+06 2% 630% 2 R338E/T343R/R403ER170E/T175R/R233E K247N/N249S/R318Y/ K82N/N84S/R150Y/R170E/ 4.1E+082.2E+08 55% 886% 4 R338E/T343R/R403E T175R/R233EY155F/K247N/N249S/R338E/ Y[155]F/K82N/N84S/R170E/ 3.7E+08 1.1E+07 3%805% 2 T343R/R403E/E410N T175R/R233E/E240N K247N/N249S/R338E/K82N/N84S/R170E/T175R/ 4.3E+08 1.2E+07 3% 930% 2 T343R/R403E/E410NR233E/E240N Y155F/K247N/N249S/ Y[155]F/K82N/N84S/R150Y/ 2.9E+08 4.1E+0714% 632% 2 R318Y/R338E R170E Y155F/K247N/N249S/ Y[155]F/K82N/N84S/R150Y/2.5E+08 9.4E+07 37% 549% 4 R318Y/T343R T175R Y155F/K247N/N249S/Y[155]F/K82N/N84S/R150Y/ 1.6E+07 5.4E+06 35% 34% 3 R318Y/R403E R233EY155F/K247N/N249S/ Y[155]F/K82N/N84S/R150Y/ 7.2E+07 2.5E+07 35% 155% 3R318Y/E410N E240N Y155F/K247N/N249S/ Y[155]F/K82N/N84S/R170E/ 1.4E+085.7E+07 41% 299% 2 R338E/R403E R233E Y155F/K247N/N249S/Y[155]F/K82N/N84S/R170E/ 7.3E+08 2.6E+08 36% 1579% 2 R338E/T343R T175RY155F/K247N/N249S/R318Y/ Y[155]F/K82N/N84S/R150Y/ 5.0E+08 2.8E+08 57%1091% 4 R338E/T343R/E410N R170E/T175R/E240N K247N/N249S/R318Y/K82N/N84S/R150Y/R170E/ 3.2E+08 1.6E+08 50% 687% 6 R338E/T343R/E410NT175R/E240N Y155F/K247N/N249S/R318Y/ Y[155]F/K82N/N84S/R150Y/ 1.6E+086.2E+07 38% 350% 2 T343R/R403E/E410N T175R/R233E/E240NK247N/N249S/R318Y/ K82N/N84S/R150Y/T175R/ 1.3E+08 3.9E+07 30% 279% 7T343R/R403E/E410N R233E/E240N Y155F/K247N/N249S/Y[155]F/K82N/N84S/R170E/ 4.7E+08 3.1E+08 66% 1009% 8 R338E/E410N E240NY155F/K247N/N249S/ Y[155]F/K82N/N84S/R150Y/ 1.3E+08 5.1E+07 40% 276% 2R318Y/T343R/R403E T175R/R233E K247N/N249S/R318Y/ K82N/N84S/R150Y/T175R/3.9E+07 2.2E+07 57% 84% 9 T343R/R403E R233E Y155F/K247N/N249S/Y[155]F/K82N/N84S/R150Y/ 3.1E+08 2.1E+08 67% 668% 4 R318Y/T343R/E410NT175R/E240N K247N/N249S/R318Y/ K82N/N84S/R150Y/T175R/ 2.0E+08 1.6E+0877% 439% 4 T343R/E410N E240N Y155F/K247N/N249S/ Y[155]F/K82N/N84S/R170E/5.9E+08 5.8E+07 10% 1269% 2 R338E/T343R/R403E T175R/R233EK247N/N249S/R338E/ K82N/N84S/R170E/T175R/ 5.6E+08 8.8E+07 16% 1215% 2T343R/R403E R233E Y155F/K247N/N249S/ Y[155]F/K82N/N84S/R170E/ 1.8E+081.1E+07 6% 391% 2 R338E/T343R/E410N T175R/E240N K247N/N249S/R338E/K82N/N84S/R170E/T175R/ 3.1E+08 1.0E+08 33% 676% 5 T343R/E410N E240NY155F/K247N/N249S/ Y[155]F/K82N/N84S/T175R/ 2.9E+08 8.8E+07 30% 635% 2T343R/R403E/E410N R233E/E240N K247N/N249S/T343R/ K82N/N84S/T175R/R233E/1.3E+08 1.7E+07 13% 285% 2 R403E/E410N E240N Y155F/R318Y/R338E/Y[155]F/R150Y/R170E/ 3.6E+08 1.5E+08 41% 771% 7 T343R T175RR318Y/R338E/T343R R150Y/R170E/T175R 1.5E+08 3.3E+07 22% 324% 2Y155F/R318Y/T343R/ Y[155]F/R150Y/T175R/ 7.1E+07 1.4E+07 20% 154% 2 R403ER233E Y155F/T343R/R403E/ Y[155]F/T175R/R233E/ 1.5E+08 2.4E+07 17% 321% 2E410N E240N Y155F/K247N/N249S/ Y[155]F/K82N/N84S/R150Y/ 3.6E+08 1.6E+0845% 772% 7 R318Y/R338E/T343R R170E/T175R K247N/N249S/R318Y/K82N/N84S/R150Y/R170E/ 3.9E+08 1.6E+08 43% 840% 4 R338E/T343R T175RY155F/K247N/N249S/ Y[155]F/K82N/N84S/T175R/ 2.8E+08 1.1E+08 38% 599% 5T343R/E410N E240N Y155F/K247N/N249S/ Y[155]F/K82N/N84S/R233E/ 2.4E+071.4E+07 59% 53% 7 R403E/E410N E240N Y155F/R338E/T343R/Y[155]F/R170E/T175R/ 3.5E+08 2.2E+08 62% 761% 6 E410N E240NR338E/T343R/E410N R170E/T175R/E240N 9.3E+07 2.8E+07 30% 201% 2Y155F/R318Y/T343R/ Y[155]F/R150Y/T175R/ 1.5E+08 6.6E+07 44% 326% 4 E410NE240N R318Y/T343R/E410N R150Y/T175R/E240N 6.2E+07 1.1E+07 17% 135% 2K228N/R318Y/R338E/ K63N/R150Y/R170E/T175R/ 2.7E+08 8.8E+07 32% 593% 3T343R/R403E/E410N R233E/E240N K228N/K247N/N249S/R318Y/K63N/K82N/N84S/R150Y/ 2.9E+08 1.3E+08 46% 636% 3 R338E/T343R/R403ER170E/T175R/R233E K228N/247N/N249S/R318Y/ K63N/K82N/N84S/R150Y/ 1.3E+084.5E+07 35% 278% 2 R338E/T343R/E410N R170E/T175R/E240NK228N/K247N/N249S/R318Y/ K63N/K82N/N84S/R150Y/ 7.1E+07 3.3E+07 46% 153%3 T343R/R403E/E410N T175R/R233E/E240N †produced in BHK-21 cells; *80%glycosylated form of E410N

TABLE 16 Catalytic activity of FIXa variants (k_(cat)) Mutation (MatureFIX Mutation (Chymotrypsin k_(cat) ±S.D. Numbering) Numbering) (s⁻¹)(s⁻¹) % CV n BeneFIX Benefix ® BeneFIX Benefix ® 2.8 1.1 39% 125Coagulation FIX (T148A) Coagulation FIX (T[148]A) Plasma Purified FIXaPlasma Purified FIXa 3.6 1.2 34% 120 Catalyst Biosciences WT CatalystBiosciences WT 3.1 1.4 46% 31 N157D N[157]D 3.3 0.5 16% 2 Y155F Y[155]F3.7 0.4 11% 2 A103N/N105S/Y155F A[103]N/N[105]S/Y[155]F 3.2 0.0  0% 2D104N/K106S/Y155F D[104]N/K[106]S/Y[155]F 2.9 0.1  4% 2 A103N/N105SA[103]N/N[105]S 3.1 1.0 31% 9 D104N/K106S D[104]N/K[106]S 3.1 1.1 34% 9K106N/V108S K[106]N/V[108]S 2.5 0.5 21% 7 D85N D[85]N 4.2 0.8 19% 15T148A T[148]A 2.2 0.9 42% 30 T148A† T[148]A† 1.6 0.2 14% 7 K5A K[5]A 3.10.2  8% 2 D64N D[64]N 1.2 0.4 31% 2 D64A D[64]A 0.3 0.2 70% 2 N167DN[167]D 2.9 0.8 27% 2 N167Q N[167]Q 2.3 0.7 32% 4 S61A S[61]A 3.6 1.541% 4 S53A S[53]A 3.7 1.7 44% 3 T159A T[159]A 3.7 1.2 34% 3 T169AT[169]A 4.6 1.6 36% 3 T172A T[172]A 4.4 1.5 34% 3 T179A T[179]A 5.1 0.612% 3 Y155H Y[155]H 4.6 0.9 18% 3 Y155Q Y[155]Q 4.4 1.0 24% 3 S158AS[158]A 3.9 0.1  3% 2 S158D S[158]D 3.5 0.3  8% 2 S158E S[158]E 3.5 0.2 5% 2 N157Q N[157]Q 3.5 0.1  4% 2 D203N/F205T D39N/F41T 1.6 0.6 40% 12D85N/D203N/F205T D[85]N/D39N/F41T 1.2 0.5 40% 5 K228N K63N 2.7 1.2 43%13 D85N/K228N D[85]N/K63N 2.7 0.8 29% 6 A103N/N105S/K228NA[103]N/N[105]S/K63N 2.1 0.5 22% 3 D104N/K106S/K228ND[104]N/K[106]S/K63N 2.4 0.1  6% 3 Y155F/K228N Y[155]F/K63N 3.3 0.3 10%2 D104N/K106S/Y155F/K228N D[104]N/K[106]S/Y[155]F/ 4.6 1.2 27% 2 K63NI251S I86S 3.8 1.1 30% 13 D85N/I251S D[85]N/I86S 2.8 0.6 22% 5D85N/D104N/K106S/I251S D[85]N/D[104]N/K[106]S/I86S 1.5 0.3 19% 5A103N/N105S/I251S A[103]N/N[105]S/I86S 2.9 1.0 36% 3 D104N/K106S/I251SD[104]N/K[106]S/I86S 2.9 0.5 18% 2 Y155F/I251S Y[155]F/I86S 3.7 0.8 22%2 A262S A95bS 2.3 0.7 32% 8 K413N K243N 2.6 0.5 19% 5 E410N E240N 5.02.2 45% 21 E410N* E240N* 2.2 0.5 25% 11 E239N E74N 1.4 0.5 36% 9T241N/H243S T76N/H78S 2.0 0.0  0% 2 K247N/N249S K82N/N84S 3.9 1.0 26% 11Y155F/K247N/N249S Y[155]F/K82N/N84S 3.3 0.7 21% 4A103N/N105S/K247N/N249S A[103]N/N[105]S/K82N/N84S 3.4 0.5 15% 6D104N/K106S/K247N/N249S D[104]N/K[106]S/K82N/N84S 3.3 1.1 32% 2D104N/K106S/Y155F/K247N/ D[104]N/K[106]S/Y[155]F/ 2.8 1.1 40% 3 N249SK82N/N84S L321N L153N 1.9 0.1  4% 2 F314N/H315S F145N/H147S No n.d. n.d.4 Activity S319N/L321S S151N/L153S 1.4 0.9 61% 3 N260S N95S 1.3 0.5 42%13 D104N/K106S/N260S D[104]N/K[106]S/N95S 1.2 0.7 58% 2 Y155F/N260SY[155]F/N95S 1.9 0.6 32% 2 D104N/K106S/Y155F/N260SD[104]N/K[106]S/Y[155]F/ 0.4 0.1 28% 2 N95S Y284N Y117N 2.0 0.9 45% 8G317N G149N No n.d. n.d. 5 Activity R318N/A320S R150N/A152S No n.d. n.d.8 Activity R318A R150A 2.4 0.8 32% 3 R318E R150E 0.6 0.2 35% 3 R318YR150Y 2.9 0.7 26% 3 R312Q R143Q 0.3 0.1 26% 3 R312A R143A 0.3 0.0  8% 2R312Y R143Y 0.4 0.3 73% 2 R312L R143L 0.7 0.3 41% 2 V202M V38M 2.6 1.037% 2 V202Y V38Y 1.8 0.2 10% 2 D203M D39M 1.8 0.8 42% 5 D203Y D39Y 1.70.5 27% 4 A204M A40M 0.6 0.5 84% 5 A204Y A40Y 1.9 0.8 42% 2 K400A/R403AK230A/R233A 0.3 0.0  5% 2 K400E/R403E K230E/R233E No n.d. n.d. 4Activity R403A R233A 0.6 0.2 24% 7 R403E R233E 0.4 0.1 25% 6 K400A K230A1.4 0.2 14% 2 K400E K230E 0.6 0.0  4% 2 K293E K126E 0.5 0.1 15% 2 K293AK126A 1.4 0.4 28% 2 R333A R165A No n.d. n.d. 2 Activity R333E R165E Non.d. n.d. 2 Activity R338A R170A 5.4 0.3  5% 2 R338E R170E 4.7 1.0 21%10 R338A/R403A R170A/R233A 3.8 0.9 24% 6 R338E/R403E R170E/R233E 3.3 1.237% 2 K293A/R403A K126A/R233A 0.4 0.0  9% 2 K293E/R403E K126E/R233E 0.10.0 37% 2 K293A/R338A/R403A K126A/R170A/R233A 1.6 0.7 41% 2K293E/R338E/R403E K126E/R170E/R233E 0.8 0.2 27% 2 R318A/R403AR150A/R233A 0.7 0.1 12% 2 R318E/R403E R150E/R233E 0.1 0.0 35% 2R318Y/E410N R150Y/E240N 3.5 0.9 27% 21 R338E/E410N R170E/E240N 5.2 0.816% 8 R338E/R403E/E410N R170E/R233E/E240N 3.3 1.3 39% 7R318Y/R338E/R403E R150Y/R170E/R233E 3.5 0.4 11% 2 D203N/F205T/K228ND39N/F41T/K63N 0.6 0.2 27% 2 D203N/F205T/E410N D39N/F41T/E240N 1.7 0.316% 6 D203N/F205T/R338E D39N/F41T/R170E 2.5 0.0  2% 2 D203N/F205T/R338AD39N/F41T/R170A 2.3 0.5 23% 3 D203N/F205T/R318Y D39N/F41T/R150Y 0.9 0.113% 4 D203N/F205T/R338E/R403E D39N/F41T/R170E/R233E 0.9 0.0  5% 2K228N/E410N K63N/E240N 3.5 0.9 27% 10 K228N/R338E K63N/R170E 4.8 0.8 17%2 K228N/R338A K63N/R170A 6.5 0.5  7% 2 K228N/R318Y K63N/R150Y 2.9 0.619% 5 K228N/R338E/R403E K63N/R170E/R233E 2.8 0.3  9% 2 R403E/E410NR233E/E240N 2.0 0.2  9% 2 R318Y/R338E/E410N R150Y/R170E/E240N 4.6 1.329% 26 D104N/K106S/R318Y/R338E/ D[104]N/K[106]S/R150Y/R170E/ 4.8 0.6 12%4 E410N E240N Y155F/R318Y/R338E/E410N Y[155]F/R150Y/R170E/E240N 5.6 1.425% 5 K228N/R318Y/E410N K63N/R150Y/E240N 5.0 0.5 10% 4 R318Y/R403E/E410NR150Y/R233E/E240N 2.3 0.3 15% 3 R318Y/R338E/R403E/E410NR150Y/R170E/R233E/E240N 5.0 3.1 63% 14 A103N/N105S/R318Y/R338E/A[103]N/N[105]S/R150Y/R170E/ 5.4 0.9 16% 5 R403E/E410N R233E/E240ND104N/K106S/R318Y/R338E/ D[104]N/K[106]S/R150Y/R170E/ 5.7 1.1 20% 3R403E/E410N R233E/E240N Y155F/R318Y/R338E/R403E/Y[155]F/R150Y/R170E/R233E/ 5.3 0.7 12% 4 E410N E240NA103N/N105S/Y155F/R318Y/ A[103]N/N[105]S/Y[155]F/ 6.4 0.5  7% 2R338E/R403E/E410N R150Y/R170E/R233E/E240N D104N/K106S/Y155F/R318Y/D[104]N/K[106]S/Y[155]F/ 8.5 0.8 10% 2 R338E/R403E/E410NR150Y/R170E/R233E/E240N D203N/F205T/R318Y/E410N D39N/F41T/R150Y/E240N1.6 0.6 36% 6 R333S R165S  0.05  0.01 22% 3 R338L R170L 9.5 1.9 21% 3K316N K148N 0.3 0.1 39% 3 K316A K148A 0.3 0.1 21% 3 K316E K148E 0.1 0.0 9% 3 K316S K148S 0.2 0.0 10% 3 K316M K148M 0.7 0.1 15% 3 E239S E74S 0.70.1 19% 3 E239A E74A 2.8 1.2 43% 3 E239R E74R 3.4 1.4 42% 3 E239K E74K3.0 1.1 36% 3 H257F H92F 3.0 1.4 46% 3 H257Y H92Y 2.0 1.1 55% 3 H257EH92E 1.3 0.4 28% 3 H257S H92S 1.8 0.3 18% 3 T412A T242A 2.6 0.3 13% 5T412V T242V 2.6 0.6 25% 8 E410N/T412A E240N/T242A 2.9 0.4 13% 4E410N/T412V E240N/T242V 2.9 0.5 16% 4 E410Q E240Q 6.0 2.8 46% 4 E410SE240S 4.9 1.6 32% 12 E410A E240A 4.8 1.6 32% 10 E410D E240D 4.0 0.7 19%4 N346D N178D 0.8 0.2 29% 4 Y155F/N346D Y[155]F/N178D 1.3 0.5 41% 2N346Y N178Y 2.6 0.2  9% 8 Y345A Y177A 0.7 0.6 83% 4 Y345T Y177T 1.3 0.327% 4 T343R T175R 4.3 1.2 27% 9 T343E T175E 1.0 0.7 72% 4 T343Q T175Q2.5 0.3 11% 3 F342I F174I 1.3 0.2 16% 3 T343R/Y345T T175R/Y177T 2.4 0.314% 3 R318Y/R338E R150Y/R170E 3.4 0.5 14% 4 Y259F/K265T/Y345TY94F/K98T/Y177T 1.7 0.1  5% 2 K228N/I251S K63N/I86S 2.7 1.1 41% 2K228N/R318Y/R338E/R403E/ K63N/R150Y/R170E/R233E/ 5.1 0.7 14% 3 E410NE240N Y155F/K228N/R318Y/R338E/ Y[155]F/K63N/R150Y/R170E/ 9.7 1.6 16% 2R403E/E410N R233E/E240N D85N/K228N/R318Y/R338E/ D[85]N/K63N/R150Y/R170E/6.0 0.6 10% 2 R403E/E410N R233E/E240N I251S/R318Y/R338E/R403E/I86S/R150Y/R170E/R233E/ 4.8 0.6 12% 4 E410N E240ND104N/K106S/I251S/R318Y/ D[104]N/K[106]S/I86S/R150Y/ 5.5 0.9 17% 8R338E/R403E/E410N R170E/R233E/E240N Y155F/I251S/R318Y/R338E/Y[155]F/I86S/R150Y/R170E/ 7.2 0.8 11% 2 R403E/E410N R233E/E240NI251S/R318Y/R338E/E410N I86S/R150Y/R170E/E240N 6.2 1.2 20% 7D104N/K106S/I251S/R318Y/ D[104]N/K[106]S/I86S/R150Y/ 3.1 0.6 19% 3R338E/E410N R170E/E240N F314N/K316S F145N/K148S 0.0 0.0  7% 2K247N/N249S/R318Y/R338E/ K82N/N84S/R150Y/R170E/ 5.8 1.1 19% 6R403E/E410N R233E/E240N Y155F/K247N/N249S/R318Y/Y[155]F/K82N/N84S/R150Y/ 6.5 1.1 17% 6 R338E/R403E/E410NR170E/R233E/E240N A103N/N105S/K247N/N249S/ A[103]N/N[105]S/K82N/N84S/4.1 0.8 18% 2 R318Y/R338E/R403E/E410N R150Y/R170E/R233E/E240ND104N/K106S/K247N/N249S/ D[104]N/K[106]S/K82N/N84S/ 5.2 0.3  6% 2R318Y/R338E/R403E/E410N R150Y/R170E/R233E/E240N K247N/N249S/R318Y/R338E/K82N/N84S/R150Y/R170E/ 3.8 1.6 41% 6 E410N E240NY155F/K247N/N249S/R318Y/ Y[155]F/K82N/N84S/R150Y/ 4.3 1.4 33% 7R338E/E410N R170E/E240N R318Y/R338E/R403E/E410S R150Y/R170E/R233E/E240S5.8 0.6 10% 4 R318Y/R338E/E410S R150Y/R170E/E240S 5.1 1.7 33% 8K228N/K247N/N249S K63N/K82N/N84S 3.5 0.1 4% 2 D104N/K106S/Y155F/K228N/D[104]N/K[106]S/Y[155]F/ 4.7 1.4 30% 2 K247N/N249S K63N/K82N/N84SD104N/K106S/K228N/K247N/ D[104]N/K[106]S/K63N/K82N/ 1.7 0.9 54% 5 N249SN84S Y155F/K228N/K247N/N249S Y[155]F/K63N/K82N/N84S 4.3 1.9 44% 2K228N/K247N/N249S/R318Y/ K63N/K82N/N84S/R150Y/R170E/ 6.1 0.7 12% 3R338E/R403E/E410N R233E/E240N R318Y/R338E/R403E/E410N/R150Y/R170E/R233E/E240N/ 7.9 2.1 26% 4 T412V T242VR318Y/R338E/R403E/E410N/ R150Y/R170E/R233E/E240N/ 8.4 1.5 18% 4 T412AT242A R318Y/R338E/R403E/T412A R150Y/R170E/R233E/T242A 5.1 1.1 21% 4R318Y/R338E/T412A R150Y/R170E/T242A 7.0 2.8 39% 6R318Y/R338E/E410N/T412V R150Y/R170E/E240N/T242V 6.3 2.3 37% 4N260S/R318Y/R338E/R403E/ N95S/R150Y/R170E/R233E/ 3.8 1.1 29% 2 E410NE240N D104N/K106S/N260S/R318Y/ D[104]N/K[106]S/N95S/R150Y/ 5.4 0.5  9% 2R338E/R403E/E410N R170E/R233E/E240N Y155F/N260S/R318Y/R338E/Y[155]F/N95S/R150Y/R170E/ 5.8 1.7 30% 2 R403E/E410N R233E/E240NR318Y/R338E/N346D/R403E/ R150Y/R170E/N178D/R233E/ 2.5 1.3 54% 2 E410NE240N Y155F/R318Y/R338E/N346D/ Y[155]F/R150Y/R170E/N178D/ 6.4 2.8 44% 2R403E/E410N R233E/E240N K247N/N249S/N260S K82N/N84S/N95S 3.3 0.3  9% 2Y155F/K247N/N249S/N260S Y[155]F/K82N/N84S/N95S 1.8 0.3 16% 2D104N/K106S/K247N/N249S/ D[104]N/K[106]S/K82N/N84S/ 0.6 0.0  7% 2 N260SN95S D104N/K106S/Y155F/K247N/ D[104]N/K[106]S/Y[155]F/ 0.5 0.0  2% 2N249S/N260S K82N/N84S/N95S K247N/N249S/N260S/R318Y/K82N/N84S/N95S/R150Y/R170E/ 6.0 0.5  9% 2 R338E/R403E/E410N R233E/E240NY155F/N260S/N346D Y[155]F/N95S/N178D 0.3 0.1 29% 2R318Y/R338E/T343R/R403E/ R150Y/R170E/T175R/R233E/ 11.8  2.4 20% 3 E410NE240N R338E/T343R R170E/T175R 7.7 1.3 17% 4 †produced in BHK-21 cells;*80% glycosylated form of E410N

TABLE 17 Catalytic activity of FIXa variants (k_(cat)) k_(cat) ±S.D.Mutation (Mature FIX Numbering) Mutation (Chymotrypsin Numbering) (s⁻¹)(s⁻¹) % CV n BeneFIX Benefix ® Coagulation FIX BeneFIX Benefix ®Coagulation FIX 2.9 1.1 39% 140 (T148A) (T[148]A) Plasma Purified FIXaPlasma Purified FIXa 3.7 1.3 36% 200 Catalyst Biosciences WT CatalystBiosciences WT 3.1 1.4 46% 33 N157D N[157]D 3.3 0.5 16% 2 Y155F Y[155]F3.7 0.4 11% 2 A103N/N105S/Y155F A[103]N/N[105]S/Y[155]F 3.2 0.0 0% 2D104N/K106S/Y155F D[104]N/K[106]S/Y[155]F 2.9 0.1 4% 2 A103N/N105SA[103]N/N[105]S 3.1 1.0 31% 9 D104N/K106S D[104]N/K[106]S 3.1 1.1 34% 9K106N/V108S K[106]N/V[108]S 2.5 0.5 21% 7 D85N D[85]N 4.1 0.8 20% 17T148A T[148]A 2.5 1.0 39% 44 T148A† T[148]A† 1.6 0.2 14% 7 K5A K[5]A 3.50.8 23% 4 D64N D[64]N 1.2 0.4 31% 2 D64A D[64]A 0.3 0.2 70% 2 N167DN[167]D 2.9 0.8 27% 2 N167Q N[167]Q 2.3 0.7 32% 4 S61A S[61]A 3.6 1.541% 4 S53A S[53]A 3.7 1.7 44% 3 T159A T[159]A 3.7 1.2 34% 3 T169AT[169]A 4.6 1.6 36% 3 T172A T[172]A 4.4 1.5 34% 3 T179A T[179]A 5.1 0.612% 3 Y155H Y[155]H 4.6 0.9 18% 3 Y155Q Y[155]Q 4.4 1.0 24% 3 S158AS[158]A 3.9 0.1 3% 2 S158D S[158]D 3.5 0.3 8% 2 S158E S[158]E 3.5 0.2 5%2 N157Q N[157]Q 3.5 0.1 4% 2 D203N/F205T D39N/F41T 1.6 0.6 40% 12D85N/D203N/F205T D[85]N/D39N/F41T 1.2 0.5 40% 5 K228N K63N 2.7 1.2 43%13 D85N/K228N D[85]N/K63N 2.7 0.8 29% 6 A103N/N105S/K228NA[103]N/N[105]S/K63N 2.1 0.5 22% 3 D104N/K106S/K228ND[104]N/K[106]S/K63N 2.4 0.1 6% 3 Y155F/K228N Y[155]F/K63N 3.3 0.3 10% 2D104N/K106S/Y155F/K228N D[104]N/K[106]S/Y[155]F/K63N 4.6 1.2 27% 2 I251SI86S 3.8 1.1 30% 13 D85N/I251S D[85]N/I86S 2.8 0.6 22% 5D85N/D104N/K106S/I251S D[85]N/D[104]N/K[106]S/I86S 1.5 0.3 19% 5A103N/N105S/I251S A[103]N/N[105]S/I86S 2.9 1.0 36% 3 D104N/K106S/I251SD[104]N/K[106]S/I86S 2.9 0.5 18% 2 Y155F/I251S Y[155]F/I86S 3.7 0.8 22%2 A262S A95bS 2.3 0.7 32% 8 K413N K243N 2.5 0.5 19% 7 E410N E240N 4.92.0 41% 27 E410N* E240N* 2.3 0.5 22% 10 E239N E74N 1.4 0.5 36% 9T241N/H243S T76N/H78S 2.0 0.0 0% 2 K247N/N249S K82N/N84S 3.9 1.0 26% 11Y155F/K247N/N249S Y[155]F/K82N/N84S 3.3 0.7 21% 4A103N/N105S/K247N/N249S A[103]N/N[105]S/K82N/N84S 3.4 0.5 15% 6D104N/K106S/K247N/N249S D[104]N/K[106]S/K82N/N84S 3.3 1.1 32% 2D104N/K106S/Y155F/K247N/N249S D[104]N/K[106]S/Y[155]F/K82N/N84S 2.8 1.140% 3 L321N L153N 1.9 0.1 4% 2 F314N/H315S F145N/H147S 0.0 0.0 7% 2K392N/K394S K222N/K224S 0.0 n.d. n.d. 0 S319N/L321S S151N/L153S 1.4 0.961% 3 N260S N95S 1.3 0.5 42% 13 D104N/K106S/N260S D[104]N/K[106]S/N95S1.2 0.7 58% 2 Y155F/N260S Y[155]F/N95S 1.9 0.6 32% 2D104N/K106S/Y155F/N260S D[104]N/K[106]S/Y[155]F/N95S 0.4 0.1 28% 2 Y284NY117N 2.0 0.9 45% 8 G317N G149N 0.0 n.d. n.d. 1 R318N/A320S R150N/A152S0.0 0.0 95% 3 R318A R150A 2.7 0.9 32% 2 R318E R150E 0.6 0.2 35% 3 R318YR150Y 2.9 0.7 26% 3 R312Q R143Q 0.3 0.1 26% 3 R312A R143A 0.3 0.0 8% 2R312Y R143Y 0.4 0.3 73% 2 R312L R143L 0.7 0.3 41% 2 V202M V38M 2.6 1.037% 2 V202Y V38Y 1.8 0.2 10% 2 D203M D39M 1.8 0.8 42% 5 D203Y D39Y 1.70.5 27% 4 A204M A40M 0.6 0.5 84% 5 A204Y A40Y 1.9 0.8 42% 2 K400A/R403AK230A/R233A 0.3 0.0 5% 2 K400E/R403E K230E/R233E 0.1 0.0 50% 3 R403AR233A 0.6 0.2 24% 7 R403E R233E 0.4 0.1 25% 6 K400A K230A 1.4 0.2 14% 2K400E K230E 0.6 0.0 4% 2 K293E K126E 0.5 0.1 15% 2 K293A K126A 1.4 0.428% 2 R333A R165A 0.1 0.0 35% 2 R333E R165E 0.0 n.d. n.d. 1 R338A R170A5.4 0.3 5% 2 R338E R170E 4.7 1.0 21% 10 R338A/R403A R170A/R233A 3.8 0.924% 6 R338E/R403E R170E/R233E 3.3 1.2 37% 2 K293A/R403A K126A/R233A 0.40.0 9% 2 K293E/R403E K126E/R233E 0.1 0.0 37% 2 K293A/R338A/R403AK126A/R170A/R233A 1.6 0.7 41% 2 K293E/R338E/R403E K126E/R170E/R233E 0.80.2 27% 2 R318A/R403A R150A/R233A 0.7 0.1 12% 2 R318E/R403E R150E/R233E0.1 0.0 35% 2 R318Y/E410N R150Y/E240N 3.5 0.9 27% 21 R338E/E410NR170E/E240N 5.2 1.1 22% 12 R338E/R403E/E410N R170E/R233E/E240N 5.8 3.052% 17 Y155F/R338E/R403E/E410N Y[155]F/R170E/R233E/E240N 5.9 0.4 7% 2R318Y/R338E/R403E R150Y/R170E/R233E 3.6 0.4 10% 3Y155F/R318Y/R338E/R403E Y[155]F/R150Y/R170E/R233E 5.1 1.0 19% 2D203N/F205T/K228N D39N/F41T/K63N 0.6 0.2 27% 2 D203N/F205T/E410ND39N/F41T/E240N 1.7 0.3 16% 6 D203N/F205T/R338E D39N/F41T/R170E 2.5 0.02% 2 D203N/F205T/R338A D39N/F41T/R170A 2.3 0.5 23% 3 D203N/F205T/R318YD39N/F41T/R150Y 0.9 0.1 13% 4 D203N/F205T/R338E/R403ED39N/F41T/R170E/R233E 0.9 0.0 5% 2 K228N/E410N K63N/E240N 3.5 0.9 27% 10K228N/R338E K63N/R170E 4.8 0.8 17% 2 K228N/R338A K63N/R170A 6.5 0.5 7% 2K228N/R318Y K63N/R150Y 2.9 0.6 19% 5 K228N/R338E/R403E K63N/R170E/R233E2.8 0.3 9% 2 R403E/E410N R233E/E240N 2.0 0.2 9% 2 R318Y/R338E/E410NR150Y/R170E/E240N 4.4 1.2 27% 42 D104N/K106S/R318Y/R338E/E410ND[104]N/K[106]S/R150Y/R170E/E240N 4.8 0.6 12% 4 Y155F/R318Y/R338E/E410NY[155]F/R150Y/R170E/E240N 5.6 1.4 25% 5 K228N/R318Y/E410NK63N/R150Y/E240N 5.0 0.5 10% 4 R318Y/R403E/E410N R150Y/R233E/E240N 2.30.3 11% 5 Y155F/R318Y/R403E/E410N Y[155]F/R150Y/R233E/E240N 2.9 0.6 20%2 R318Y/R338E/R403E/E410N R150Y/R170E/R233E/E240N 5.8 2.8 48% 26A103N/N105S/R318Y/R338E/R403E/ A[103]N/N[105]S/R150Y/R170E/R233E/ 5.40.9 16% 5 E410N E240N D104N/K106S/R318Y/R338E/R403E/D[104]N/K[106]S/R150Y/R170E/R233E/ 5.7 1.1 20% 3 E410N E240NY155F/R318Y/R338E/R403E/E410N Y[155]F/R150Y/R170E/R233E/E240N 5.3 0.712% 4 A103N/N105S/Y155F/R318Y/R338E/A[103]N/N[105]S/Y[155]F/R150Y/R170E/ 6.4 0.5 7% 2 R403E/E410NR233E/E240N D104N/K106S/Y155F/R318Y/R338E/D[104]N/K[106]S/Y[155]F/R150Y/R170E/ 8.5 0.8 10% 2 R403E/E410NR233E/E240N D203N/F205T/R318Y/E410N D39N/F41T/R150Y/E240N 1.6 0.6 36% 6R333S R165S 0.1 0.0 22% 3 R338L R170L 9.5 1.9 21% 3 K316N K148N 0.3 0.139% 3 K316A K148A 0.3 0.1 21% 3 K316E K148E 0.1 0.0 9% 3 K316S K148S 0.20.0 10% 3 K316M K148M 0.7 0.1 15% 3 E239S E74S 0.7 0.1 19% 3 E239A E74A2.8 1.2 43% 3 E239R E74R 3.4 1.4 42% 3 E239K E74K 3.0 1.1 36% 3 H257FH92F 3.0 1.4 46% 3 H257Y H92Y 2.0 1.1 55% 3 H257E H92E 1.3 0.4 28% 3H257S H92S 1.8 0.3 18% 3 T412A T242A 2.6 0.3 13% 5 T412V T242V 2.6 0.625% 8 E410N/T412A E240N/T242A 2.9 0.4 13% 4 E410N/T412V E240N/T242V 2.90.5 16% 4 E410Q E240Q 6.0 2.8 46% 4 E410S E240S 4.9 1.6 32% 12 E410AE240A 4.8 1.6 32% 10 E410D E240D 4.0 0.7 19% 4 N346D N178D 0.8 0.2 29% 4Y155F/N346D Y[155]F/N178D 1.3 0.5 41% 2 N346Y N178Y 2.6 0.2 9% 8 Y345AY177A 0.7 0.6 83% 4 Y345T Y177T 1.3 0.3 27% 4 T343R T175R 4.1 1.1 27% 12T343E T175E 1.0 0.7 72% 4 T343Q T175Q 2.5 0.3 11% 3 F342I F174I 1.3 0.216% 3 T343R/Y345T T175R/Y177T 2.4 0.3 14% 3 R318Y/R338E R150Y/R170E 3.40.5 14% 4 Y259F/K265T/Y345T Y94F/K98T/Y177T 1.7 0.1 5% 2 K228N/I251SK63N/I86S 2.7 1.1 41% 2 K228N/R318Y/R338E/R403E/E410NK63N/R150Y/R170E/R233E/E240N 5.1 0.7 14% 3Y155F/K228N/R318Y/R338E/R403E/ Y[155]F/K63N/R150Y/R170E/R233E/E240N 6.73.0 45% 5 E410N D85N/K228N/R318Y/R338E/R403E/D[85]N/K63N/R150Y/R170E/R233E/E240N 6.0 0.6 10% 2 E410NI251S/R318Y/R338E/R403E/E410N I86S/R150Y/R170E/R233E/E240N 4.8 0.6 12% 4D104N/K106S/I251S/R318Y/R338E/ D[104]N/K[106]S/I86S/R150Y/R170E/ 5.5 0.917% 8 R403E/E410N R233E/E240N Y155F/I251S/R318Y/R338E/R403E/D[104]N/K[106]S/I86S/R150Y/R170E/ 7.2 0.8 11% 2 E410N R233E/E240NI251S/R318Y/R338E/E410N I86S/R150Y/R170E/E240N 6.4 2.0 31% 10D104N/K106S/I251S/R318Y/R338E/ D[104]N/K[106]S/I86S/R150Y/R170E/ 3.1 0.619% 3 E410N E240N F314N/K316S F145N/K148S 0.0 0.0 7% 2K247N/N249S/R318Y/R338E/R403E/ K82N/N84S/R150Y/R170E/R233E/E240N 5.8 1.119% 6 E410N Y155F/K247N/N249S/R318Y/R338E/Y[155]F/K82N/N84S/R150Y/R170E/R233E/ 6.2 1.0 16% 10 R403E/E410N E240NA103N/N105S/K247N/N249S/R318Y/ A[103]N/N[105]S/K82N/N84S/R150Y/ 3.9 0.411% 6 R338E/R403E/E410N R170E/R233E/E240N D104N/K106S/K247N/N249S/R318Y/D[104]N/K[106]S/K82N/N84S/R150Y/ 5.2 0.3 6% 2 R338E/R403E/E410NR170E/R233E/E240N D104N/K106S/Y155F/K247N/N249S/D[104]N/K[106]S/Y[155]F/K82N/N84S/ 6.9 4.7 67% 6 R318Y/R338E/R403E/E410NR150Y/R170E/R233E/E240N K247N/N249S/R318Y/R338E/E410NK82N/N84S/R150Y/R170E/E240N 3.8 1.6 41% 6 Y155F/K247N/N249S/R318Y/R338E/Y[155]F/K82N/N84S/R150Y/R170E/E240N 4.5 1.3 28% 9 E410NR318Y/R338E/R403E/E410S R150Y/R170E/R233E/E240S 7.4 2.3 31% 7R318Y/R338E/E410S R150Y/R170E/E240S 5.1 1.7 33% 8 K228N/K247N/N249SK63N/K82N/N84S 3.5 0.1 4% 2 D104N/K106S/Y155F/K228N/K247N/D[104]N/K[106]S/Y[155]F/K63N/K82N/ 4.7 1.4 30% 2 N249S N84SD104N/K106S/K228N/K247N/N249S D[104]N/K[106]S/K63N/K82N/N84S 1.7 0.9 54%5 Y155F/K228N/K247N/N249S Y[155]F/K63N/K82N/N84S 4.3 1.9 44% 2K228N/K247N/N249S/R318Y/R338E/ K63N/K82N/N84S/R150Y/R170E/R233E/ 7.1 2.231% 17 R403E/E410N E240N D104N/K106S/K228N/K247N/N249S/D[104]N/K[106]S/K63N/K82N/N84S/R150Y/ 6.1 3.7 61% 7R318Y/R338E/R403E/E410N R170E/R233E/E240N Y155F/K228N/K247N/N249S/R318Y/Y[155]F/K63N/K82N/N84S/R150Y/R170E/ 5.1 1.8 34% 5 R338E/R403E/E410NR233E/E240N R318Y/R338E/R403E/E410N/T412V R150Y/R170E/R233E/E240N/T242V7.0 2.1 30% 6 R318Y/R338E/R403E/E410N/T412AR150Y/R170E/R233E/E240N/T242A 7.8 1.6 20% 6 R318Y/R338E/R403E/T412AR150Y/R170E/R233E/T242A 5.1 1.1 21% 4 R318Y/R338E/T412AR150Y/R170E/T242A 7.0 2.8 39% 6 R318Y/R338E/E410N/T412VR150Y/R170E/E240N/T242V 5.2 1.7 33% 11 N260S/R318Y/R338E/R403E/E410NN95S/R150Y/R170E/R233E/E240N 3.8 1.1 29% 2D104N/K106S/N260S/R318Y/R338E/ D[104]N/K[106]S/N95S/R150Y/R170E/ 5.4 0.59% 2 R403E/E410N R233E/E240N Y155F/N260S/R318Y/R338E/R403E/Y[155]F/N95S/R150Y/R170E/R233E/E240N 5.8 1.7 30% 2 E410NR318Y/R338E/N346D/R403E/E410N R150Y/R170E/N178D/R233E/E240N 2.5 1.3 54%2 Y155F/R318Y/R338E/N346D/R403E/ Y[155]F/R150Y/R170E/N178D/R233E/ 6.42.8 44% 2 E410N E240N K247N/N249S/N260S K82N/N84S/N95S 3.3 0.3 9% 2Y155F/K247N/N249S/N260S Y[155]F/K82N/N84S/N95S 1.8 0.3 16% 2D104N/K106S/K247N/N249S/N260S D[104]N/K[106]S/K82N/N84S/N95S 0.6 0.0 7%2 D104N/K106S/Y155F/K247N/N249S/ D[104]N/K[106]S/Y[155]F/K82N/N84S/ 0.50.0 2% 2 N260S N95S K247N/N249S/N260S/R318Y/R338E/K82N/N84S/N95S/R150Y/R170E/R233E/ 3.4 2.1 62% 6 R403E/E410N E240NY155F/K247N/N249S/N260S/R318Y/ Y[155]F/K82N/N84S/N95S/R150Y/R170E/ 3.61.2 33% 5 R338E/R403E/E410N R233E/E240N Y155F/N260S/N346DY[155]F/N95S/N178D 0.3 0.1 29% 2 R318Y/R338E/T343R/R403E/E410NR150Y/R170E/T175R/R233E/E240N 9.7 2.6 27% 13Y155F/R318Y/R338E/T343R/R403E/ Y[155]F/R150Y/R170E/T175R/R233E/ 7.8 1.924% 4 E410N E240N D104N/K106S/R318Y/R338E/T343R/D[104]N/K[106]S/R150Y/R170E/T175R/ 5.7 2.3 41% 5 R403E/E410N R233E/E240NR338E/T343R R170E/T175R 7.1 1.4 20% 7 T343R/N346Y T175R/N178Y 2.3 0.521% 11 R318Y/R338E/N346Y/R403E/E410N R150Y/R170E/N178Y/R233E/E240N 3.40.3 9% 3 R318Y/R338E/T343R/N346Y/R403E/R150Y/R170E/T175R/N178Y/R233E/E240N 4.6 1.2 26% 5 E410N T343R/N346DT175R/N178D 1.9 0.4 21% 2 R318Y/R338E/T343R/N346D/R403E/R150Y/R170E/T175R/N178D/R233E/E240N 5.4 2.0 36% 2 E410NR318Y/R338E/Y345A/R403E/E410N R150Y/R170E/Y177A/R233E/E240N 1.4 0.5 36%6 R318Y/R338E/Y345A/N346D/R403E/ R150Y/R170E/Y177A/N178D/R233E/E240N 1.20.3 26% 3 E410N Y155F/K247N/N249S/R318Y/R338E/Y[155]F/K82N/N84S/R150Y/R170E/R233E 5.7 3.2 55% 5 R403EK247N/N249S/R318Y/R338E/R403E K82N/N84S/R150Y/R170E/R233E 10.5 3.6 34% 2Y155F/K247N/N249S/R318Y/R403E/ Y[155]F/K82N/N84S/R150Y/R233E/E240N 1.20.5 40% 3 E410N K247N/N249S/R318Y/R403E/E410NK82N/N84S/R150Y/R233E/E240N 2.9 1.6 55% 10Y155F/K247N/N249S/R338E/R403E/ Y[155]F/K82N/N84S/R170E/R233E/E240N 5.00.6 13% 3 E410N K247N/N249S/R338E/R403E/E410NK82N/N84S/R170E/R233E/E240N 4.8 0.8 17% 2 R318Y/R338E/T343R/R403ER150Y/R170E/T175R/R233E 6.7 1.6 24% 4 Y155F/R318Y/R338E/T343R/R403EY[155]F/R150Y/R170E/T175R/R233E 8.2 4.1 50% 4 R318Y/R338E/T343R/E410NR150Y/R170E/T175R/E240N 4.9 1.2 24% 16 Y155F/R318Y/R338E/T343R/E410NY[155]F/R150Y/R170E/T175R/E240N 9.2 3.1 33% 4 R318Y/T343R/R403E/E410NR150Y/T175R/R233E/E240N 5.3 0.9 17% 3 Y155F/R318Y/T343R/R403E/E410NY[155]F/R150Y/T175R/R233E/E240N 8.8 0.3 4% 2 R338E/T343R/R403E/E410NR170E/T175R/R233E/E240N 9.8 1.4 15% 2 Y155F/R338E/T343R/R403E/E410NY[155]F/R170E/T175R/R233E/E240N 5.7 1.1 20% 4Y155F/K247N/N249S/R318Y/R338E/ Y[155]F/K82N/N84S/R150Y/R170E/T175R/ 9.73.4 35% 11 T343R/R403E/E410N R233E/E240N K247N/N249S/R318Y/R338E/T343R/K82N/N84S/R150Y/R170E/T175R/R233E/ 10.6 3.6 34% 5 R403E/E410N E240NK228N/I251S/R318Y/R338E/R403E/ K63N/I86S/R150Y/R170E/R233E/E240N 7.5 3.344% 7 E410N Y155F/K228N/I251S/R318Y/R338E/Y[155]F/K63N/I86S/R150Y/R170E/R233E/ 5.3 1.9 36% 5 R403E/E410N E240NN260S/R318Y/R338E/T343R/R403E/ N95S/R150Y/R170E/T175R/R233E/E240N 8.93.6 40% 7 E410N Y155F/N260S/R318Y/R338E/T343R/Y[155]F/N95S/R150Y/R170E/T175R/R233E/ 5.8 1.6 28% 5 R403E/E410N E240NK228N/K247N/N249S/R318Y/R338E/ K63N/K82N/N84S/R150Y/R170E/T175R/ 9.9 3.232% 12 T343R/R403E/E410N R233E/E240N Y155F/K228N/K247N/N249S/R318Y/Y[155]F/K63N/K82N/N84S/R150Y/R170E/ 9.4 2.3 25% 5R338E/T343R/R403E/E410N T175R/R233E/E240N Y155F/R338E/T343R/R403EY[155]F/R170E/T175R/R233E 5.2 0.9 18% 5 R338E/T343R/R403ER170E/T175R/R233E 6.9 0.3 4% 2 Y155F/R338E/T343R/R403E/E410SY[155]F/R170E/T175R/R233E/E240S 6.8 2.4 34% 6Y155F/N260S/R338E/T343R/R403E Y[155]F/N95S/R170E/T175R/R233E 6.4 3.8 59%6 Y155F/I251S/R338E/T343R/R403E Y[155]F/I86S/R170E/T175R/R233E 5.9 0.712% 2 R318Y/R338E/T343R/R403E/E410S R150Y/R170E/T175R/R233E/E240S 7.61.7 22% 14 Y155F/K247N/N249S/T343R/R403E Y[155]F/K82N/N84S/T175R/R233E4.7 0.2 5% 4 Y155F/K247N/N249S/R318Y/R338E/Y[155]F/K82N/N84S/R150Y/R170E/T175R/ 10.6 0.8 8% 2 T343R/R403E R233EK247N/N249S/R318Y/R338E/T343R/ K82N/N84S/R150Y/R170E/T175R/R233E 9.2 3.336% 4 R403E Y155F/K247N/N249S/R338E/T343R/Y[155]F/K82N/N84S/R170E/T175R/R233E/ 9.8 0.7 8% 2 R403E/E410N E240NK247N/N249S/R338E/T343R/R403E/ K82N/N84S/R170E/T175R/R233E/E240N 10.81.6 15% 2 E410N Y155F/K247N/N249S/R318Y/R338EY[155]F/K82N/N84S/R150Y/R170E 7.5 1.5 20% 2Y155F/K247N/N249S/R318Y/T343R Y[155]F/K82N/N84S/R150Y/T175R 10.3 3.3 32%4 Y155F/K247N/N249S/R318Y/R403E Y[155]F/K82N/N84S/R150Y/R233E 1.7 0.742% 3 Y155F/K247N/N249S/R318Y/E410N Y[155]F/K82N/N84S/R150Y/E240N 3.40.9 26% 3 Y155F/K247N/N249S/R338E/R403E Y[155]F/K82N/N84S/R170E/R233E5.3 0.6 11% 2 Y155F/K247N/N249S/R338E/T343RY[155]F/K82N/N84S/R170E/T175R 10.6 1.1 10% 2Y155F/K247N/N249S/R318Y/R338E/ Y[155]F/K82N/N84S/R150Y/R170E/T175R/ 7.72.3 30% 4 T343R/E410N E240N K247N/N249S/R318Y/R338E/T343R/K82N/N84S/R150Y/R170E/T175R/E240N 8.8 4.4 50% 6 E410NY155F/K247N/N249S/R318Y/T343R/ Y[155]F/K82N/N84S/R150Y/T175R/R233E/ 9.00.4 5% 2 R403E/E410N E240N K247N/N249S/R318Y/T343R/R403E/K82N/N84S/R150Y/T175R/R233E/E240N 9.5 1.6 17% 7 E410NY155F/K247N/N249S/R338E/E410N Y[155]F/K82N/N84S/R170E/E240N 7.3 3.5 48%8 Y155F/K247N/N249S/R318Y/T343R/ Y[155]F/K82N/N84S/R150Y/T175R/R233E 7.52.1 28% 2 R403E K247N/N249S/R318Y/T343R/R403EK82N/N84S/R150Y/T175R/R233E 3.7 1.6 44% 9 Y155F/K247N/N249S/R318Y/T343R/Y[155]F/K82N/N84S/R150Y/T175R/E240N 8.1 4.1 51% 4 E410NK247N/N249S/R318Y/T343R/E410N K82N/N84S/R150Y/T175R/E240N 6.1 2.6 42% 4Y155F/K247N/N249S/R338E/T343R/ Y[155]F/K82N/N84S/R170E/T175R/R233E 14.60.2 1% 2 R403E K247N/N249S/R338E/T343R/R403E K82N/N84S/R170E/T175R/R233E14.6 0.4 3% 2 Y155F/K247N/N249S/R338E/T343R/Y[155]F/K82N/N84S/R170E/T175R/E240N 4.8 1.0 20% 2 E410NK247N/N249S/R338E/T343R/E410N K82N/N84S/R170E/T175R/E240N 7.9 1.4 18% 5Y155F/K247N/N249S/T343R/R403E/ Y[155]F/K82N/N84S/T175R/R233E/E240N 15.03.0 20% 2 E410N K247N/N249S/T343R/R403E/E410NK82N/N84S/T175R/R233E/E240N 8.0 2.8 36% 2 Y155F/R318Y/R338E/T343RY[155]F/R150Y/R170E/T175R 7.9 3.0 38% 7 R318Y/R338E/T343RR150Y/R170E/T175R 4.5 1.2 27% 2 Y155F/R318Y/T343R/R403EY[155]F/R150Y/T175R/R233E 5.0 1.1 22% 2 Y155F/T343R/R403E/E410NY[155]F/T175R/R233E/E240N 6.6 1.4 21% 2 Y155F/K247N/N249S/R318Y/R338E/Y[155]F/K82N/N84S/R150Y/R170E/T175R 8.5 3.3 39% 7 T343RK247N/N249S/R318Y/R338E/T343R K82N/N84S/R150Y/R170E/T175R 8.0 1.7 22% 4Y155F/K247N/N249S/T343R/E410N Y[155]F/K82N/N84S/T175R/E240N 7.9 1.6 20%5 Y155F/K247N/N249S/R403E/E410N Y[155]F/K82N/N84S/R233E/E240N 2.7 1.452% 7 Y155F/R338E/T343R/E410N Y[155]F/R170E/T175R/E240N 6.0 2.2 37% 6R338E/T343R/E410N R170E/T175R/E240N 3.1 0.5 16% 2Y155F/R318Y/T343R/E410N Y[155]F/R150Y/T175R/E240N 5.0 1.3 25% 4R318Y/T343R/E410N R150Y/T175R/E240N 3.2 0.4 13% 2K228N/R318Y/R338E/T343R/R403E/ K63N/R150Y/R170E/T175R/R233E/E240N 10.50.7 6% 3 E410N K228N/K247N/N249S/R318Y/R338E/K63N/K82N/N84S/R150Y/R170E/T175R/ 10.9 1.4 13% 3 T343R/R403E R233EK228N/K247N/N249S/R318Y/R338E/ K63N/K82N/N84S/R150Y/R170E/T175R/ 4.7 0.48% 2 T343R/E410N E240N K228N/K247N/N249S/R318Y/T343R/K63N/K82N/N84S/R150Y/T175R/R233E/ 8.1 2.1 26% 3 R403E/E410N E240N†produced in BHK-21 cells; *80% glycosylated form of E410N

TABLE 18 Catalytic activity of FIXa variants (K_(M)) Mutation (MatureFIX Mutation (Chymotrypsin K_(M) ±S.D. Numbering) Numbering) (nM) (nM) %CV n BeneFIX Benefix ® Coagulation BeneFIX Benefix ® 76.9 27.5 36% 125FIX (T148A) Coagulation FIX (T[148]A) Plasma Purified FIXa PlasmaPurified FIXa 74.5 25.5 34% 120 Catalyst Biosciences WT CatalystBiosciences WT 74.7 23.1 31% 31 N157D N[157]D 121.8 53.0 44% 2 Y155FY[155]F 90.3 10.3 11% 2 A103N/N105S/Y155F A[103]N/N[105]S/Y[155]F 80.42.5 3% 2 D104N/K106S/Y155F D[104]N/K[106]S/Y[155]F 81.5 5.2 6% 2A103N/N105S A[103]N/N[105]S 88.0 22.5 26% 9 D104N/K106S D[104]N/K[106]S83.2 18.2 22% 9 K106N/V108S K[106]N/V[108]S 91.9 20.2 22% 7 D85N D[85]N64.5 23.1 36% 15 T148A T[148]A 64.5 25.1 39% 30 T148A† T[148]A† 74.616.1 22% 7 K5A K[5]A 55.0 0.3 1% 2 D64N D[64]N 121.4 58.8 48% 2 D64AD[64]A 129.4 36.3 28% 2 N167D N[167]D 94.6 7.0 7% 2 N167Q N[167]Q 77.135.8 46% 4 S61A S[61]A 84.6 35.6 42% 4 S53A S[53]A 109.9 11.6 11% 3T159A T[159]A 100.9 1.2 1% 3 T169A T[169]A 99.7 10.8 11% 3 T172A T[172]A96.2 22.1 23% 3 T179A T[179]A 94.5 16.7 18% 3 Y155H Y[155]H 93.9 15.817% 3 Y155Q Y[155]Q 87.6 29.8 34% 3 S158A S[158]A 107.7 0.4 0% 2 S158DS[158]D 87.0 9.0 10% 2 S158E S[158]E 96.0 14.1 15% 2 N157Q N[157]Q 107.85.5 5% 2 D203N/F205T D39N/F41T 74.3 19.5 26% 12 D85N/D203N/F205TD[85]N/D39N/F41T 40.6 9.1 22% 5 K228N K63N 72.5 25.5 35% 13 D85N/K228ND[85]N/K63N 60.1 13.4 22% 6 A103N/N105S/K228N A[103]N/N[105]S/K63N 76.515.8 21% 3 D104N/K106S/K228N D[104]N/K[106]S/K63N 96.8 21.2 22% 3Y155F/K228N Y[155]F/K63N 73.7 3.7 5% 2 D104N/K106S/Y155F/K228ND[104]N/K[106]S/Y[155]F/K63N 76.2 6.4 8% 2 I251S I86S 64.3 13.3 21% 13D85N/I251S D[85]N/I86S 51.5 15.3 30% 5 D85N/D104N/K106S/I251SD[85]N/D[104]N/K[106]S/I86S 46.4 19.0 41% 5 A103N/N105S/I251SA[103]N/N[105]S/I86S 90.9 41.2 45% 3 D104N/K106S/I251SD[104]N/K[106]S/I86S 97.5 13.8 14% 2 Y155F/I251S Y[155]F/I86S 56.4 17.531% 2 A262S A95bS 99.2 19.9 20% 8 K413N K243N 109.6 41.0 37% 5 E410NE240N 46.2 21.5 47% 21 E410N* E240N* 83.3 36.9 44% 11 E239N E74N 78.329.5 38% 9 T241N/H243S T76N/H78S 104.5 3.5 3% 2 K247N/N249S K82N/N84S75.0 15.4 21% 11 Y155F/K247N/N249S Y[155]F/K82N/N84S 67.1 23.6 35% 4A103N/N105S/K247N/N249S A[103]N/N[105]S/K82N/N84S 84.0 9.7 12% 6D104N/K106S/K247N/N249S D[104]N/K[106]S/K82N/N84S 102.3 23.0 23% 2D104N/K106S/Y155F/K247N/N249S D[104]N/K[106]S/Y[155]F/K82N 89.3 10.3 12%3 N84S L321N L153N 118.5 10.6 9% 2 F314N/H315S F145N/H147S No n.d. n.d.4 Activity S319N/L321S S151N/L153S 54.2 14.8 27% 3 N260S N95S 83.4 27.533% 13 D104N/K106S/N260S D[104]N/K[106]S/N95S 94.3 6.8 7% 2 Y155F/N260SY[155]F/N95S 130.6 78.1 60% 2 D104N/K106S/Y155F/N260SD[104]N/K[106]S/Y[155]F/N95S 107.7 74.8 69% 2 Y284N Y117N 59.8 23.5 39%8 G317N G149N No n.d. n.d. 5 Activity R318N/A320S R150N/A152S No n.d.n.d. 8 Activity R318A R150A 52.8 25.8 49% 3 R318E R150E 33.6 10.3 31% 3R318Y R150Y 40.7 7.6 19% 3 R312Q R143Q 29.9 5.0 17% 3 R312A R143A 61.616.9 27% 2 R312Y R143Y 27.2 11.4 42% 2 R312L R143L 28.8 0.6 2% 2 V202MV38M 40.2 1.0 2% 2 V202Y V38Y 70.6 2.3 3% 2 D203M D39M 40.6 7.9 19% 5D203Y D39Y 58.0 19.5 34% 4 A204M A40M 34.0 9.2 27% 5 A204Y A40Y 39.510.3 26% 2 K400A/R403A K230A/R233A 56.7 10.0 18% 2 K400E/R403EK230E/R233E No n.d. n.d. 4 Activity R403A R233A 46.4 5.2 11% 7 R403ER233E 67.0 19.4 29% 6 K400A K230A 74.6 22.1 30% 2 K400E K230E 61.3 9.315% 2 K293E K126E 63.2 13.9 22% 2 K293A K126A 73.7 35.2 48% 2 R333AR165A No n.d. n.d. 2 Activity R333E R165E No n.d. n.d. 2 Activity R338AR170A 33.7 3.7 11% 2 R338E R170E 28.7 9.0 31% 10 R338A/R403A R170A/R233A73.6 18.1 25% 6 R338E/R403E R170E/R233E 51.9 11.9 23% 2 K293A/R403AK126A/R233A 69.2 10.2 15% 2 K293E/R403E K126E/R233E 104.1 31.0 30% 2K293A/R338A/R403A K126A/R170A/R233A 65.4 1.3 2% 2 K293E/R338E/R403EK126E/R170E/R233E 50.0 15.1 30% 2 R318A/R403A R150A/R233A 45.7 1.6 3% 2R318E/R403E R150E/R233E 75.3 47.7 63% 2 R318Y/E410N R150Y/E240N 49.614.3 29% 21 R338E/E410N R170E/E240N 12.6 4.2 33% 8 R338E/R403E/E410NR170E/R233E/E240N 45.5 12.8 28% 7 R318Y/R338E/R403E R150Y/R170E/R233E53.7 1.9 4% 2 D203N/F205T/K228N D39N/F41T/K63N 39.9 3.8 9% 2D203N/F205T/E410N D39N/F41T/E240N 45.5 12.0 26% 6 D203N/F205T/R338ED39N/F41T/R170E 24.1 5.6 23% 2 D203N/F205T/R338A D39N/F41T/R170A 38.59.9 26% 3 D203N/F205T/R318Y D39N/F41T/R150Y 47.5 6.4 13% 4D203N/F205T/R338E/R403E D39N/F41T/R170E/R233E 51.1 10.7 21% 2K228N/E410N K63N/E240N 44.3 13.0 29% 10 K228N/R338E K63N/R170E 23.1 3.013% 2 K228N/R338A K63N/R170A 31.2 4.5 14% 2 K228N/R318Y K63N/R150Y 61.35.4 9% 5 K228N/R338E/R403E K63N/R170E/R233E 59.2 4.9 8% 2 R403E/E410NR233E/E240N 93.7 1.0 1% 2 R318Y/R338E/E410N R150V/R170E/E240N 14.2 4.330% 26 D104N/K106S/R318Y/R338E/E410N D[104]N/K[106]S/R150Y/R170E/ 18.94.1 22% 4 E240N Y155F/R318Y/R338E/E410N Y[155]F/R150Y/R170E/E240N 16.04.8 30% 5 K228N/R318Y/E410N K63N/R150Y/E240N 42.0 4.7 11% 4R318Y/R403E/E410N R150Y/R233E/E240N 88.3 12.4 14% 3R318Y/R338E/R403E/E410N R150Y/R170E/R233E/E240N 45.5 12.2 27% 14A103N/N105S/R318Y/R338E/R403E/ A[103]N/N[105]S/R150Y/R170E/ 44.7 20.947% 5 E410N R233E/E240N D104N/K106S/R318Y/R338E/R403E/D[104]N/K[106]S/R150Y/R170E/ 38.5 16.1 42% 3 E410N R233E/E240NY155F/R318Y/R338E/R403E/E410N Y[155]F/R150Y/R170E/R233E/ 30.4 10.5 35% 4E240N A103N/N105S/Y155F/R318Y/R338E/ A[103]N/N[105]S/Y[155]F/R150Y/ 50.74.5 9% 2 R403E/E410N R170E/R233E/E240N D104N/K106S/Y155F/R318Y/R338E/D[104]N/K[106]S/Y[155]F/R150Y/ 48.0 2.1 4% 2 R403E/E410NR170E/R233E/E240N D203N/F205T/R318Y/E410N D39N/F41T/R150Y/E240N 45.713.4 29% 6 R333S R165S 605.9 317.5 52% 3 R338L R170L 47.9 9.0 19% 3K316N K148N 62.5 15.6 25% 3 K316A K148A 55.2 4.1 7% 3 K316E K148E 110.525.1 23% 3 K316S K148S 57.3 4.6 8% 3 K316M K148M 26.0 16.7 64% 3 E239SE74S 28.5 19.2 67% 3 E239A E74A 55.4 18.4 33% 3 E239R E74R 58.3 13.9 24%3 E239K E74K 59.2 25.5 43% 3 H257F H92F 62.0 30.1 49% 3 H257Y H92Y 59.325.0 42% 3 H257E H92E 59.7 39.6 66% 3 H257S H92S 56.0 24.7 44% 3 T412AT242A 76.1 44.7 59% 5 T412V T242V 51.2 18.9 37% 8 E410N/T412AE240N/T242A 37.2 3.6 10% 4 E410N/T412V E240N/T242V 33.3 4.9 15% 4 E410QE240Q 56.1 18.0 32% 4 E410S E240S 50.0 11.9 24% 12 E410A E240A 47.7 11.724% 10 E410D E240D 71.9 26.9 37% 4 N346D N178D 45.7 7.8 17% 4Y155F/N346D Y[155]F/N178D 104.4 14.5 14% 2 N346Y N178Y 27.4 4.2 15% 8Y345A Y177A 50.8 32.4 64% 4 Y345T Y177T 28.6 7.9 28% 4 T343R T175R 31.310.9 35% 9 T343E T175E 27.3 10.0 37% 4 T343Q T175Q 37.0 9.1 25% 3 F342IF174I 30.0 19.1 64% 3 T343R/Y345T T175R/Y177T 26.5 6.8 26% 3 R318Y/R338ER150Y/R170E 24.6 5.5 22% 4 Y259F/K265T/Y345T Y94F/K98T/Y177T 30.9 4.816% 2 K228N/I251S K63N/I86S 122.6 53.5 44% 2K228N/R318Y/R338E/R403E/E410N K63N/R150Y/R170E/R233E/ 36.1 14.0 39% 3E240N Y155F/K228N/R318Y/R338E/R403E/ Y[155]F/K63N/R150Y/R170E/ 48.0 9.821% 2 E410N R233E/E240N D85N/K228N/R318Y/R338E/R403E/D[85]N/K63N/R150Y/R170E/ 39.3 9.8 25% 2 E410N R233E/E240NI251S/R318Y/R338E/R403E/E410N I86S/R150Y/R170E/R233E/E240N 33.4 10.2 30%4 D104N/K106S/I251S/R318Y/R338E/ D[104]N/K[106]S/I86S/R150Y/ 46.2 7.717% 8 R403E/E410N R170E/R233E/E240N Y155F/I251S/R318Y/R338E/R403E/Y[155]F/I86S/R150Y/R170E/ 43.3 7.0 16% 2 E410N R233E/E240NI251S/R318Y/R338E/E410N I86S/R150Y/R170E/E240N 16.2 1.8 11% 7D104N/K106S/I251S/R318Y/R338E/ D[104]N/K[106]S/I86S/R150Y/ 24.3 8.6 35%3 E410N R170E/E240N F314N/K316S F145N/K148S 635.1 569.9 90% 2K247N/N249S/R318Y/R338E/R403E/ K82N/N84S/R150Y/R170E/R233E/ 39.2 8.3 21%6 E410N E240N Y155F/K247N/N249S/R318Y/R338E/ Y[155]F/K82N/N84S/R150Y/39.1 14.7 38% 6 R403E/E410N R170E/R233E/E240NA103N/N105S/K247N/N249S/R318Y/ A[103]N/N[105]S/K82N/N84S/ 39.7 4.5 11% 2R338E/R403E/E410N R150Y/R170E/R233E/E240N D104N/K106S/K247N/N249S/R318Y/D[104]N/K[106]S/K82N/N84S/ 59.0 0.6 1% 2 R338E/R403E/E410NR150Y/R170E/R233E/E240N K247N/N249S/R318Y/R338E/E410NK82N/N84S/R150Y/R170E/E240N 16.6 3.7 22% 6Y155F/K247N/N249S/R318Y/R338E/ Y[155]F/K82N/N84S/R150Y/ 15.3 4.1 27% 7E410N R170E/E240N R318Y/R338E/R403E/E410S R150Y/R170E/R233E/E240S 35.112.4 35% 4 R318Y/R338E/E410S R150Y/R170E/E240S 16.4 4.0 25% 8K228N/K247N/N249S K63N/K82N/N84S 94.5 27.0 29% 2D104N/K106S/Y155F/K228N/K247N/ D[104]N/K[106]S/Y[155]F/K63N/ 75.3 26.435% 2 N249S K82N/N84S D104N/K106S/K228N/K247N/N249SD[104]N/K[106]S/K63N/K82N/ 77.1 18.3 24% 5 N84S Y155F/K228N/K247N/N249SY[155]F/K63N/K82N/N84S 79.2 27.6 35% 2 K228N/K247N/N249S/R318Y/R338E/K63N/K82N/N84S/R150Y/R170E/ 55.8 15.8 28% 3 R403E/E410N R233E/E240NR318Y/R338E/R403E/E410N/T412V R150Y/R170E/R233E/E240N/ 44.3 19.2 43% 4T242V R318Y/R338E/R403E/E410N/T412A R150Y/R170E/R233E/E240N/ 33.5 4.814% 4 T242A R318Y/R338E/R403E/T412A R150Y/R170E/R233E/T242A 67.5 11.617% 4 R318Y/R338E/T412A R150Y/R170E/T242A 23.5 5.3 22% 6R318Y/R338E/E410N/T412V R150Y/R170E/E240N/T242V 29.7 10.9 37% 4N260S/R318Y/R338E/R403E/E410N N95S/R150Y/R170E/R233E/ 72.4 20.2 28% 2E240N D104N/K106S/N260S/R318Y/R338E/ D[104]N/K[106]S/N95S/R150Y/ 61.10.0 0% 2 R403E/E410N R170E/R233E/E240N Y155F/N260S/R318Y/R338E/R403E/Y[155]F/N95S/R150Y/R170E/ 83.9 4.4 5% 2 E410N R233E/E240NR318Y/R338E/N346D/R403E/E410N R150Y/R170E/N178D/R233E/ 77.7 20.9 27% 2E240N Y155F/R318Y/R338E/N346D/R403E/ Y[155]F/R150Y/R170E/N178D/ 100.015.6 16% 2 E410N R233E/E240N K247N/N249S/N260S K82N/N84S/N95S 114.1 0.00% 2 Y155F/K247N/N249S/N260S Y[155]F/K82N/N84S/N95S 96.5 5.5 6% 2D104N/K106S/K247N/N249S/N260S D[104]N/K[106]S/K82N/N84S/ 61.2 14.1 23% 2N95S D104N/K106S/Y155F/K247N/N249S/ D[104]N/K[106]S/Y[155]F/K82N/ 68.533.2 49% 2 N260S N84S/N95S K247N/N249S/N260S/R318Y/R338E/K82N/N84S/N95S/R150Y/R170E/ 62.2 0.0 0% 2 R403E/E410N R233E/E240NY155F/N260S/N346D Y[155]F/N95S/N178D 127.9 6.2 5% 2R318Y/R338E/T343R/R403E/E410N R150Y/R170E/T175R/R233E/ 22.3 5.0 23% 3E240N R338E/T343R R170E/T175R 13.6 3.7 27% 4 †produced in BHK-21 cells;*80% glycosylated form of E410N

TABLE 19 Catalytic activity of FIXa variants (K_(M)) Mutation (MatureFIX Mutation (Chymotrypsin K_(M) ±S.D. Numbering) Numbering) (nM) (nM) %CV n BeneFIX Benefix ® Coagulation FIX BeneFIX Benefix ® Coagulation FIX75.8 27.2 36% 140 (T148A) (T[148]A) Plasma Purified FIXa Plasma PurifiedFIXa 73.3 26.8 37% 200 Catalyst Biosciences WT Catalyst Biosciences WT72.3 24.3 34% 33 N157D N[157]D 121.8 53.0 44% 2 Y155F Y[155]F 90.3 10.311% 2 A103N/N105S/Y155F A[103]N/N[105]S/Y[155]F 80.4 2.5 3% 2D104N/K106S/Y155F D[104]N/K[106]S/Y[155]F 81.5 5.2 6% 2 A103N/N105SA[103]N/N[105]S 88.0 22.5 26% 9 D104N/K106S D[104]N/K[106]S 83.2 18.222% 9 K106N/V108S K[106]N/V[108]S 91.9 20.2 22% 7 D85N D[85]N 64.5 21.934% 17 T148A T[148]A 70.1 26.9 38% 44 T148A† T[148]A† 74.6 16.1 22% 7K5A K[5]A 65.4 26.8 41% 4 D64N D[64]N 121.4 58.8 48% 2 D64A D[64]A 129.436.3 28% 2 N167D N[167]D 94.6 7.0 7% 2 N167Q N[167]Q 77.1 35.8 46% 4S61A S[61]A 84.6 35.6 42% 4 S53A S[53]A 109.9 11.6 11% 3 T159A T[159]A100.9 1.2 1% 3 T169A T[169]A 99.7 10.8 11% 3 T172A T[172]A 96.2 22.1 23%3 T179A T[179]A 94.5 16.7 18% 3 Y155H Y[155]H 93.9 15.8 17% 3 Y155QY[155]Q 87.6 29.8 34% 3 S158A S[158]A 107.7 0.4 0% 2 S158D S[158]D 87.09.0 10% 2 S158E S[158]E 96.0 14.1 15% 2 N157Q N[157]Q 107.8 5.5 5% 2D203N/F205T D39N/F41T 74.3 19.5 26% 12 D85N/D203N/F205T D[85]N/D39N/F41T40.6 9.1 22% 5 K228N K63N 72.5 25.5 35% 13 D85N/K228N D[85]N/K63N 60.113.4 22% 6 A103N/N105S/K228N A[103]N/N[105]S/K63N 76.5 15.8 21% 3D104N/K106S/K228N D[104]N/K[106]S/K63N 96.8 21.2 22% 3 Y155F/K228NY[155]F/K63N 73.7 3.7 5% 2 D104N/K106S/Y155F/K228ND[104]N/K[106]S/Y[155]F/K63N 76.2 6.4 8% 2 I251S I86S 64.3 13.3 21% 13D85N/I251S D[85]N/I86S 51.5 15.3 30% 5 D85N/D104N/K106S/I251SD[85]N/D[104]N/K[106]S/I86S 46.4 19.0 41% 5 A103N/N105S/I251SA[103]N/N[105]S/I86S 90.9 41.2 45% 3 D104N/K106S/I251SD[104]N/K[106]S/I86S 97.5 13.8 14% 2 Y155F/I251S Y[155]F/I86S 56.4 17.531% 2 A262S A95bS 99.2 19.9 20% 8 K413N K243N 106.3 40.4 38% 7 E410NE240N 45.9 19.1 42% 27 E410N* E240N* 85.2 38.1 45% 10 E239N E74N 78.329.5 38% 9 T241N/H243S T76N/H78S 104.5 3.5 3% 2 K247N/N249S K82N/N84S75.0 15.4 21% 11 Y155F/K247N/N249S Y[155]F/K82N/N84S 67.1 23.6 35% 4A103N/N105S/K247N/N249S A[103]N/N[105]S/K82N/N84S 84.0 9.7 12% 6D104N/K106S/K247N/N249S D[104]N/K[106]S/K82N/N84S 102.3 23.0 23% 2D104N/K106S/Y155F/K247N/N249S D[104]N/K[106]S/Y[155]F/K82N/N84S 89.310.3 12% 3 L321N L153N 118.5 10.6 9% 2 F314N/H315S F145N/H147S 93.0 14.315% 2 K392N/K394S K222N/K224S 0.0 n.d. n.d. 0 S319N/L321S S151N/L153S54.2 14.8 27% 3 N260S N95S 83.4 27.5 33% 13 D104N/K106S/N260SD[104]N/K[106]S/N95S 94.3 6.8 7% 2 Y155F/N260S Y[155]F/N95S 130.6 78.160% 2 D104N/K106S/Y155F/N260S D[104]N/K[106]S/Y[155]F/N95S 107.7 74.869% 2 Y284N Y117N 59.8 23.5 39% 8 G317N G149N 104.6 n.d. n.d. 1R318N/A320S R150N/A152S 84.5 21.2 25% 3 R318A R150A 62.3 28.2 45% 2R318E R150E 33.6 10.3 31% 3 R318Y R150Y 40.7 7.6 19% 3 R312Q R143Q 29.95.0 17% 3 R312A R143A 61.6 16.9 27% 2 R312Y R143Y 27.2 11.4 42% 2 R312LR143L 28.8 0.6 2% 2 V202M V38M 40.2 1.0 2% 2 V202Y V38Y 70.6 2.3 3% 2D203M D39M 40.6 7.9 19% 5 D203Y D39Y 58.0 19.5 34% 4 A204M A40M 34.0 9.227% 5 A204Y A40Y 39.5 10.3 26% 2 K400A/R403A K230A/R233A 56.7 10.0 18% 2K400E/R403E K230E/R233E 137.1 68.4 50% 3 R403A R233A 46.4 5.2 11% 7R403E R233E 67.0 19.4 29% 6 K400A K230A 74.6 22.1 30% 2 K400E K230E 61.39.3 15% 2 K293E K126E 63.2 13.9 22% 2 K293A K126A 73.7 35.2 48% 2 R333AR165A 406.7 117.5 29% 2 R333E R165E 437.3 n.d. n.d. 1 R338A R170A 33.73.7 11% 2 R338E R170E 28.7 9.0 31% 10 R338A/R403A R170A/R233A 73.6 18.125% 6 R338E/R403E R170E/R233E 51.9 11.9 23% 2 K293A/R403A K126A/R233A69.2 10.2 15% 2 K293E/R403E K126E/R233E 104.1 31.0 30% 2K293A/R338A/R403A K126A/R170A/R233A 65.4 1.3 2% 2 K293E/R338E/R403EK126E/R170E/R233E 50.0 15.1 30% 2 R318A/R403A R150A/R233A 45.7 1.6 3% 2R318E/R403E R150E/R233E 75.3 47.7 63% 2 R318Y/E410N R150Y/E240N 49.614.3 29% 21 R338E/E410N R170E/E240N 12.6 3.5 28% 12 R338E/R403E/E410NR170E/R233E/E240N 36.7 12.2 33% 17 Y155F/R338E/R403E/E410NY[155]F/R170E/R233E/E240N 33.6 8.6 26% 2 R318Y/R338E/R403ER150Y/R170E/R233E 59.7 10.4 17% 3 Y155F/R318Y/R338E/R403EY[155]F/R150Y/R170E/R233E 67.1 27.9 42% 2 D203N/F205T/K228ND39N/F41T/K63N 39.9 3.8 9% 2 D203N/F205T/E410N D39N/F41T/E240N 45.5 12.026% 6 D203N/F205T/R338E D39N/F41T/R170E 24.1 5.6 23% 2 D203N/F205T/R338AD39N/F41T/R170A 38.5 9.9 26% 3 D203N/F205T/R318Y D39N/F41T/R150Y 47.56.4 13% 4 D203N/F205T/R338E/R403E D39N/F41T/R170E/R233E 51.1 10.7 21% 2K228N/E410N K63N/E240N 44.3 13.0 29% 10 K228N/R338E K63N/R170E 23.1 3.013% 2 K228N/R338A K63N/R170A 31.2 4.5 14% 2 K228N/R318Y K63N/R150Y 61.35.4 9% 5 K228N/R338E/R403E K63N/R170E/R233E 59.2 4.9 8% 2 R403E/E410NR233E/E240N 93.7 1.0 1% 2 R318Y/R338E/E410N R150Y/R170E/E240N 13.9 4.029% 42 D104N/K106S/R318Y/R338E/E410N D[104]N/K[106]S/R150Y/R170E/E240N18.9 4.1 22% 4 Y155F/R318Y/R338E/E410N Y[155]F/R150Y/R170E/E240N 16.04.8 30% 5 K228N/R318Y/E410N K63N/R150Y/E240N 42.0 4.7 11% 4R318Y/R403E/E410N R150Y/R233E/E240N 94.2 21.1 22% 5Y155F/R318Y/R403E/E410N Y[155]F/R150Y/R233E/E240N 111.4 74.7 67% 2R318Y/R338E/R403E/E410N R150Y/R170E/R233E/E240N 43.2 13.8 32% 26A103N/N105S/R318Y/R338E/R403E/ A[103]N/N[105]S/R150Y/R170E/R233E/ 44.720.9 47% 5 E410N E240N D104N/K106S/R318Y/R338E/R403E/D[104]N/K[106]S/R150Y/R170E/R233E/ 38.5 16.1 42% 3 E410N E240NY155F/R318Y/R338E/R403E/E410N Y[155]F/R150Y/R170E/R233E/E240N 30.4 10.535% 4 A103N/N105S/Y155F/R318Y/R338E/A[103]N/N[105]S/Y[155]F/R150Y/R170E/ 50.7 4.5 9% 2 R403E/E410NR233E/E240N D104N/K106S/Y155F/R318Y/R338E/D[104]N/K[106]S/Y[155]F/R150Y/R170E/ 48.0 2.1 4% 2 R403E/E410NR233E/E240N D203N/F205T/R318Y/E410N D39N/F41T/R150Y/E240N 45.7 13.4 29%6 R333S R165S 605.9 317.5 52% 3 R338L R170L 47.9 9.0 19% 3 K316N K148N62.5 15.6 25% 3 K316A K148A 55.2 4.1 7% 3 K316E K148E 110.5 25.1 23% 3K316S K148S 57.3 4.6 8% 3 K316M K148M 26.0 16.7 64% 3 E239S E74S 28.519.2 67% 3 E239A E74A 55.4 18.4 33% 3 E239R E74R 58.3 13.9 24% 3 E239KE74K 59.2 25.5 43% 3 H257F H92F 62.0 30.1 49% 3 H257Y H92Y 59.3 25.0 42%3 H257E H92E 59.7 39.6 66% 3 H257S H92S 56.0 24.7 44% 3 T412A T242A 76.144.7 59% 5 T412V T242V 51.2 18.9 37% 8 E410N/T412A E240N/T242A 37.2 3.610% 4 E410N/T412V E240N/T242V 33.3 4.9 15% 4 E410Q E240Q 56.1 18.0 32% 4E410S E240S 50.0 11.9 24% 12 E410A E240A 47.7 11.7 24% 10 E410D E240D71.9 26.9 37% 4 N346D N178D 45.7 7.8 17% 4 Y155F/N346D Y[155]F/N178D104.4 14.5 14% 2 N346Y N178Y 27.4 4.2 15% 8 Y345A Y177A 50.8 32.4 64% 4Y345T Y177T 28.6 7.9 28% 4 T343R T175R 34.5 11.8 34% 12 T343E T175E 27.310.0 37% 4 T343Q T175Q 37.0 9.1 25% 3 F342I F174I 30.0 19.1 64% 3T343R/Y345T T175R/Y177T 26.5 6.8 26% 3 R318Y/R338E R150Y/R170E 24.6 5.522% 4 Y259F/K265T/Y345T Y94F/K98T/Y177T 30.9 4.8 16% 2 K228N/I251SK63N/I86S 122.6 53.5 44% 2 K228N/R318Y/R338E/R403E/E410NK63N/R150Y/R170E/R233E/E240N 36.1 14.0 39% 3Y155F/K228N/R318Y/R338E/R403E/ Y[155]F/K63N/R150Y/R170E/R233E/ 40.8 15.037% 5 E410N E240N D85N/K228N/R318Y/R338E/R403E/D[85]N/K63N/R150Y/R170E/R233E/ 39.3 9.8 25% 2 E410N E240NI251S/R318Y/R338E/R403E/E410N I86S/R150Y/R170E/R233E/E240N 33.4 10.2 30%4 D104N/K106S/I251S/R318Y/R338E/ D[104]N/K[106]S/I86S/R150Y/R170E/ 46.27.7 17% 8 R403E/E410N R233E/E240N Y155F/I251S/R318Y/R338E/R403E/D[104]N/K[106]S/I86S/R150Y/R170E/ 43.3 7.0 16% 2 E410N R233E/E240NI251S/R318Y/R338E/E410N I86S/R150Y/R170E/E240N 16.1 2.7 17% 10D104N/K106S/I251S/R318Y/R338E/ D[104]N/K[106]S/I86S/R150Y/R170E/ 24.38.6 35% 3 E410N E240N F314N/K316S F145N/K148S 635.1 569.9 90% 2K247N/N249S/R318Y/R338E/R403E/ K82N/N84S/R150Y/R170E/R233E/E240N 39.28.3 21% 6 E410N Y155F/K247N/N249S/R318Y/R338E/Y[155]F/K82N/N84S/R150Y/R170E/ 36.3 12.8 35% 10 R403E/E410N R233E/E240NA103N/N105S/K247N/N249S/R318Y/ A[103]N/N[105]S/K82N/N84S/R150Y/ 28.0 9.534% 6 R338E/R403E/E410N R170E/R233E/E240N D104N/K106S/K247N/N249S/R318Y/D[104]N/K[106]S/K82N/N84S/R150Y/ 59.0 0.6 1% 2 R338E/R403E/E410NR170E/R233E/E240N D104N/K106S/Y155F/K247N/N249S/D[104]N/K[106]S/Y[155]F/K82N/N84S/ 51.8 16.7 32% 6R318Y/R338E/R403E/E410N R150Y/R170E/R233E/E240NK247N/N249S/R318Y/R338E/E410N K82N/N84S/R150Y/R170E/E240N 16.6 3.7 22% 6Y155F/K247N/N249S/R318Y/R338E/ Y[155]F/K82N/N84S/R150Y/R170E/ 14.7 3.927% 9 E410N E240N R318Y/R338E/R403E/E410S R150Y/R170E/R233E/E240S 36.49.5 26% 7 R318Y/R338E/E410S R150Y/R170E/E240S 16.4 4.0 25% 8K228N/K247N/N249S K63N/K82N/N84S 94.5 27.0 29% 2D104N/K106S/Y155F/K228N/K247N/ D[104]N/K[106]S/Y[155]F/K63N/K82N/ 75.326.4 35% 2 N249S N84S D104N/K106S/K228N/K247N/N249SD[104]N/K[106]S/K63N/K82N/N84S 77.1 18.3 24% 5 Y155F/K228N/K247N/N249SY[155]F/K63N/K82N/N84S 79.2 27.6 35% 2 K228N/K247N/N249S/R318Y/R338E/K63N/K82N/N84S/R150Y/R170E/R233E/ 49.7 15.6 31% 17 R403E/E410N E240ND104N/K106S/K228N/K247N/N249S/ D[104]N/K[106]S/K63N/K82N/N84S/ 53.3 12.223% 7 R318Y/R338E/R403E/E410N R150Y/R170E/R233E/E240NY155F/K228N/K247N/N249S/R318Y/ Y[155]F/K63N/K82N/N84S/R150Y/R170E/ 45.417.7 39% 5 R338E/R403E/E410N R233E/E240N R318Y/R338E/R403E/E410N/T412VR150Y/R170E/R233E/E240N/T242V 48.3 16.2 33% 6R318Y/R338E/R403E/E410N/T412A R150Y/R170E/R233E/E240N/T242A 34.4 10.029% 6 R318Y/R338E/R403E/T412A R150Y/R170E/R233E/T242A 67.5 11.6 17% 4R318Y/R338E/T412A R150Y/R170E/T242A 23.5 5.3 22% 6R318Y/R338E/E410N/T412V R150Y/R170E/E240N/T242V 23.6 12.3 52% 11N260S/R318Y/R338E/R403E/E410N N95S/R150Y/R170E/R233E/E240N 72.4 20.2 28%2 D104N/K106S/N260S/R318Y/R338E/ D[104]N/K[106]S/N95S/R150Y/R170E/ 61.10.0 0% 2 R403E/E410N R233E/E240N Y155F/N260S/R318Y/R338E/R403E/Y[155]F/N95S/R150Y/R170E/R233E/ 83.9 4.4 5% 2 E410N E240NR318Y/R338E/N346D/R403E/E410N R150Y/R170E/N178D/R233E/E240N 77.7 20.927% 2 Y155F/R318Y/R338E/N346D/R403E/ Y[155]F/R150Y/R170E/N178D/R233E/100.0 15.6 16% 2 E410N E240N K247N/N249S/N260S K82N/N84S/N95S 114.1 0.00% 2 Y155F/K247N/N249S/N260S Y[155]F/K82N/N84S/N95S 96.5 5.5 6% 2D104N/K106S/K247N/N249S/N260S D[104]N/K[106]S/K82N/N84S/N95S 61.2 14.123% 2 D104N/K106S/Y155F/K247N/N249S/ D[104]N/K[106]S/Y[155]F/K82N/N84S/68.5 33.2 49% 2 N260S N95S K247N/N249S/N260S/R318Y/R338E/K82N/N84S/N95S/R150Y/R170E/R233E/ 47.4 12.1 26% 6 R403E/E410N E240NY155F/K247N/N249S/N260S/R318Y/ Y[155]F/K82N/N84S/N95S/R150Y/R170E/ 95.473.0 77% 5 R338E/R403E/E410N R233E/E240N Y155F/N260S/N346DY[155]F/N95S/N178D 127.9 6.2 5% 2 R318Y/R338E/T343R/R403E/E410NR150Y/R170E/T175R/R233E/E240N 24.7 7.2 29% 13Y155F/R318Y/R338E/T343R/R403E/ Y[155]F/R150Y/R170E/T175R/R233E/ 27.2 5.721% 4 E410N E240N D104N/K106S/R318Y/R338E/T343R/D[104]N/K[106]S/R150Y/R170E/T175R/ 26.6 5.0 19% 5 R403E/E410NR233E/E240N R338E/T343R R170E/T175R 14.3 3.6 25% 7 T343R/N346YT175R/N178Y 26.0 7.3 28% 11 R318Y/R338E/N346Y/R403E/E410NR150Y/R170E/N178Y/R233E/E240N 28.1 7.5 27% 3R318Y/R338E/T343R/N346Y/R403E/ R150Y/R170E/T175R/N178Y/R233E/ 15.8 4.025% 5 E410N E240N T343R/N346D T175R/N178D 118.5 42.9 36% 2R318Y/R338E/T343R/N346D/R403E/ R150Y/R170E/T175R/N178D/R233E/ 67.0 26.840% 2 E410N E240N R318Y/R338E/Y345A/R403E/E410NR150Y/R170E/Y177A/R233E/E240N 18.8 8.8 47% 6R318Y/R338E/Y345A/N346D/R403E/ R150Y/R170E/Y177A/N178D/R233E/ 56.5 16.128% 3 E410N E240N Y155F/K247N/N249S/R318Y/R338E/Y[155]F/K82N/N84S/R150Y/R170E/ 67.3 17.7 26% 5 R403E R233EK247N/N249S/R318Y/R338E/R403E K82N/N84S/R150Y/R170E/R233E 53.6 22.1 41%2 Y155F/K247N/N249S/R318Y/R403E/ Y[155]F/K82N/N84S/R150Y/R233E/ 125.49.1 7% 3 E410N E240N K247N/N249S/R318Y/R403E/E410NK82N/N84S/R150Y/R233E/E240N 110.9 29.5 27% 10Y155F/K247N/N249S/R338E/R403E/ Y[155]F/K82N/N84S/R170E/R233E/E240N 48.711.4 23% 3 E410N K247N/N249S/R338E/R403E/E410NK82N/N84S/R170E/R233E/E240N 25.0 7.9 31% 2 R318Y/R338E/T343R/R403ER150Y/R170E/T175R/R233E 44.3 11.0 25% 4 Y155F/R318Y/R338E/T343R/R403EY[155]F/R150Y/R170E/T175R/R233E 34.0 8.7 26% 4 R318Y/R338E/T343R/E410NR150Y/R170E/T175R/E240N 16.4 5.9 36% 16 Y155F/R318Y/R338E/T343R/E410NY[155]F/R150Y/R170E/T175R/E240N 25.6 5.4 21% 4 R318Y/T343R/R403E/E410NR150Y/T175R/R233E/E240N 93.9 14.0 15% 3 Y155F/R318Y/T343R/R403E/E410NY[155]F/R150Y/T175R/R233E/E240N 34.0 7.7 23% 2 R338E/T343R/R403E/E410NR170E/T175R/R233E/E240N 34.7 14.3 41% 2 Y155F/R338E/T343R/R403E/E410NY[155]F/R170E/T175R/R233E/E240N 25.9 8.2 32% 4Y155F/K247N/N249S/R318Y/R338E/ Y[155]F/K82N/N84S/R150Y/R170E/ 25.7 8.433% 11 T343R/R403E/E410N T175R/R233E/E240NK247N/N249S/R318Y/R338E/T343R/ K82N/N84S/R150Y/R170E/T175R/R233E/ 29.27.9 27% 5 R403E/E410N E240N K228N/I251S/R318Y/R338E/R403E/K63N/I86S/R150Y/R170E/R233E/E240N 36.4 10.8 30% 7 E410NY155F/K228N/I251S/R318Y/R338E/ Y[155]F/K63N/I86S/R150Y/R170E/R233E/ 39.37.3 19% 5 R403E/E410N E240N N260S/R318Y/R338E/T343R/R403E/N95S/R150Y/R170E/T175R/R233E/E240N 32.1 10.3 32% 7 E410NY155F/N260S/R318Y/R338E/T343R/ Y[155]F/N95S/R150Y/R170E/T175R/ 40.2 11.629% 5 R403E/E410N R233E/E240N K228N/K247N/N249S/R318Y/R338E/K63N/K82N/N84S/R150Y/R170E/T175R/ 25.1 5.4 21% 12 T343R/R403E/E410NR233E/E240N Y155F/K228N/K247N/N249S/R318Y/Y[155]F/K63N/K82N/N84S/R150Y/R170E/ 36.8 18.8 51% 5R338E/T343R/R403E/E410N T175R/R233E/E240N Y155F/R338E/T343R/R403EY[155]F/R170E/T175R/R233E 28.9 9.1 31% 5 R338E/T343R/R403ER170E/T175R/R233E 23.5 6.5 28% 2 Y155F/R338E/T343R/R403E/E410SY[155]F/R170E/T175R/R233E/E240S 23.9 3.1 13% 6Y155F/N260S/R338E/T343R/R403E Y[155]F/N95S/R170E/T175R/R233E 69.2 27.840% 6 Y155F/I251S/R338E/T343R/R403E Y[155]F/I86S/R170E/T175R/R233E 19.63.4 17% 2 R318Y/R338E/T343R/R403E/E410S R150Y/R170E/T175R/R233E/E240S19.0 6.4 33% 14 Y155F/K247N/N249S/T343R/R403EY[155]F/K82N/N84S/T175R/R233E 59.6 20.3 34% 4Y155F/K247N/N249S/R318Y/R338E/ Y[155]F/K82N/N84S/R150Y/R170E/ 36.5 3.510% 2 T343R/R403E T175R/R233E K247N/N249S/R318Y/R338E/T343R/K82N/N84S/R150Y/R170E/T175R/R233E 28.4 17.8 63% 4 R403EY155F/K247N/N249S/R338E/T343R/ Y[155]F/K82N/N84S/R170E/T175R/R233E/ 26.41.3 5% 2 R403E/E410N E240N K247N/N249S/R338E/T343R/R403E/K82N/N84S/R170E/T175R/R233E/E240N 25.1 3.0 12% 2 E410NY155F/K247N/N249S/R318Y/R338E Y[155]F/K82N/N84S/R150Y/R170E 26.3 8.8 33%2 Y155F/K247N/N249S/R318Y/T343R Y[155]F/K82N/N84S/R150Y/T175R 42.1 12.830% 4 Y155F/K247N/N249S/R318Y/R403E Y[155]F/K82N/N84S/R150Y/R233E 108.622.3 21% 3 Y155F/K247N/N249S/R318Y/E410N Y[155]F/K82N/N84S/R150Y/E240N48.8 12.8 26% 3 Y155F/K247N/N249S/R338E/R403EY[155]F/K82N/N84S/R170E/R233E 40.9 12.9 31% 2Y155F/K247N/N249S/R338E/T343R Y[155]F/K82N/N84S/R170E/T175R 15.3 4.0 26%2 Y155F/K247N/N249S/R318Y/R338E/ Y[155]F/K82N/N84S/R150Y/R170E/ 17.7 6.034% 4 T343R/E410N T175R/E240N K247N/N249S/R318Y/R338E/T343R/K82N/N84S/R150Y/R170E/T175R/E240N 32.8 22.9 70% 6 E410NY155F/K247N/N249S/R318Y/T343R/ Y[155]F/K82N/N84S/R150Y/T175R/ 60.6 26.043% 2 R403E/E410N R233E/E240N K247N/N249S/R318Y/T343R/R403E/K82N/N84S/R150Y/T175R/R233E/E240N 80.5 31.3 39% 7 E410NY155F/K247N/N249S/R338E/E410N Y[155]F/K82N/N84S/R170E/E240N 17.7 7.6 43%8 Y155F/K247N/N249S/R318Y/T343R/ Y[155]F/K82N/N84S/R150Y/T175R/ 60.5 7.512% 2 R403E R233E K247N/N249S/R318Y/T343R/R403EK82N/N84S/R150Y/T175R/R233E 105.3 25.8 25% 9Y155F/K247N/N249S/R318Y/T343R/ Y[155]F/K82N/N84S/R150Y/T175R/ 38.1 29.678% 4 E410N E240N K247N/N249S/R318Y/T343R/E410NK82N/N84S/R150Y/T175R/E240N 40.1 25.9 64% 4Y155F/K247N/N249S/R338E/T343R/ Y[155]F/K82N/N84S/R170E/T175R/R233E 25.12.8 11% 2 R403E K247N/N249S/R338E/T343R/R403EK82N/N84S/R170E/T175R/R233E 26.3 3.5 13% 2Y155F/K247N/N249S/R338E/T343R/ Y[155]F/K82N/N84S/R170E/T175R/E240N 27.07.1 26% 2 E410N K247N/N249S/R338E/T343R/E410NK82N/N84S/R170E/T175R/E240N 27.5 11.1 40% 5Y155F/K247N/N249S/T343R/R403E/ Y[155]F/K82N/N84S/T175R/R233E/E240N 52.05.4 10% 2 E410N K247N/N249S/T343R/R403E/E410NK82N/N84S/T175R/R233E/E240N 60.0 13.9 23% 2 Y155F/R318Y/R338E/T343RY[155]F/R150Y/R170E/T175R 24.2 8.8 36% 7 R318Y/R338E/T343RR150Y/R170E/T175R 30.0 1.5 5% 2 Y155F/R318Y/T343R/R403EY[155]F/R150Y/T175R/R233E 72.7 29.5 41% 2 Y155F/T343R/R403E/E410NY[155]F/T175R/R233E/E240N 44.6 1.9 4% 2 Y155F/K247N/N249S/R318Y/R338E/Y[155]F/K82N/N84S/R150Y/R170E/ 27.6 13.2 48% 7 T343R T175RK247N/N249S/R318Y/R338E/T343R K82N/N84S/R150Y/R170E/T175R 24.4 13.5 55%4 Y155F/K247N/N249S/T343R/E410N Y[155]F/K82N/N84S/T175R/E240N 34.4 20.058% 5 Y155F/K247N/N249S/R403E/E410N Y[155]F/K82N/N84S/R233E/E240N 131.353.1 40% 7 Y155F/R338E/T343R/E410N Y[155]F/R170E/T175R/E240N 22.4 13.862% 6 R338E/T343R/E410N R170E/T175R/E240N 35.5 15.9 45% 2Y155F/R318Y/T343R/E410N Y[155]F/R150Y/T175R/E240N 40.3 22.9 57% 4R318Y/T343R/E410N R150Y/T175R/E240N 52.3 2.4 5% 2K228N/R318Y/R338E/T343R/R403E/ K63N/R150Y/R170E/T175R/R233E/E240N 40.39.6 24% 3 E410N K228N/K247N/N249S/R318Y/R338E/K63N/K82N/N84S/R150Y/R170E/T175R/ 44.4 23.7 53% 3 T343R/R403E R233EK228N/247N/N249S/R318Y/R338E/ K63N/K82N/N84S/R150Y/R170E/T175R/ 38.110.4 27% 2 T343R/E410N E240N K228N/K247N/N249S/R318Y/T343R/K63N/K82N/N84S/R150Y/T175R/R233E/ 125.1 36.4 29% 3 R403E/E410N E240N†produced in BHK-21 cells; *80% glycosylated form of E410N

Example 5 Determination of the Inhibition of FIXa by theAntithrombin/Heparin Complex

Inhibition of wild-type FIXa or FIXa variants by theAntithrombin/heparin complex (AT-III/heparin) was assessed by measuringthe level of inhibition by various concentrations of AT-III/heparin onthe catalytic activity of FIXa towards a small molecule substrate,Mesyl-D-CHG-Gly-Arg-AMC (Pefafluor FIXa; Pentapharm). A K_(0.5) value isdetermined for each FIXa variant tested, which corresponds to the molarconcentration of AT-III that was required for 50% inhibition (IC₅₀) ofthe catalytic activity of a FIXa variant under the predefined conditionsof the assay Inhibition reactions were performed in the presence of lowmolecular weight heparin (LMWH; Calbiochem) or full-lengthunfractionated heparin (UFH; Calbiochem), the latter requiring modifiedprotocol conditions to account for an increase in the rate ofinhibition. The apparent second-order rate constant (k_(app)) for theinhibition of wild-type FIXa or FIXa variants by the AT-III/UFH complexwas also directly evaluated using a modified protocol, in which the timeof incubation with the AT-III/UFH complex was varied.

A. Inhibition of FIXa by the Antithrombin/LMWH Complex

For inhibition reactions in the presence of LMWH, a 200 nM solution ofAT-III/LMWH (final 2 μM LMWH) was prepared by dilution of a 20 μM stockof plasma purified human AT-III (Molecular Innovations) into a solutionof 2 μM LMWH in a 1.2 mL volume of 1× Buffer A (50 mM Tris, 100 mM NaCl,10 mM CaCl₂, 0.01% Tween-20, pH 7.4). This solution of AT-III/LMWH wasfor use as the highest concentration in the assay. AT-III/LMWH solutionswere incubated for at least 30 minutes at room temperature and thenserially diluted 1.5-fold in a 96 deep-well polypropylene plate with afinal volume of 400 μL 1× Buffer A that contained 2 μM LMWH, resultingin dilutions of 200 nM, 133.3, nM 88.9 nM, 59.3 nM, 39.5 nM, 26.3 nM,17.6 nM and 0 nM (i.e. rows A-H). A total of 25 μL was aliquoted intotheir respective rows of a 96-well V-bottom storage plate to fill allcolumns (i.e. 1-12). FIXa variants were initially diluted to 100 nM in1× Buffer A. Subsequently, 36 μL of each 100 nM FIXa variant was dilutedto a concentration of 1.8 nM in 2.0 mL of 1× Buffer A and then 60 μL ofthis solution was aliquoted into a 96-well V-bottom storage plateaccording to a predefined plate map (4 FIXa variants per plate).

Assay reactions were initiated using a BioMek FX liquid handling systemprogrammed to dispense 25 μL of the FIXa solutions into the platescontaining 25 μL of each dilution of AT-III/LMWH per well for a total oftwo duplicate assay plates for each FIXa variant. The final inhibitionassay conditions were: 0.9 nM FIXa and AT-III dilutions ranging from 0to 100 nM in 1 μM LMWH Inhibition reactions were further incubated for 1minute at room temperature (˜25° C.) before a 25 μL aliquot of thereaction was transferred by the BioMek FX to a 96-well black half-areaplate containing 25 μL of 1.6 mM Mesyl-D-CHG-Gly-Arg-AMC per well inassay Buffer B (50 mM Tris, 100 mM NaCl, 10 mM CaCl₂, 0.01% Tween-20, pH7.4, 60% ethylene glycol). Polybrene (hexadimethrine bromide) at a finalconcentration of 5 mg/mL was added in Buffer B to quench the AT-III/LMWHreaction. Residual activity of FIXa was assessed by following theinitial rates of substrate cleavage for 60 minutes in a fluorescencereader set to 25° C. The final assay conditions for determination ofresidual activity are 0.45 nM FIXa variant, 0.8 mMMesyl-D-CHG-Gly-Arg-AMC, 30% ethylene glycol and 5 mg/mL polybrene in 50mM Tris, 100 mM NaCl, 10 mM CaCl₂, 0.01% Tween-20, pH 7.4.

To determine the degree of inhibition by AT-III/LMWH for FIXa or FIXavariants, raw data collected with the SoftMax Pro application (MolecularDevices) were exported as .XML files. Further non-linear data analyseswere performed with XLfit4, a software package for automated curvefitting and statistical analysis within the Microsoft Excel spreadsheetenvironment (IDBS Software) or directly within the ActivityBase softwarepackage using the XE Runner data analysis module (IDBS Software). Thetemplate was used to calculate the AT-III dilution series, ratio ofAT-III to FIXa, and the Vi/Vo ratios for each FIXa replicate at eachexperimental AT-III concentration. The spreadsheet template was used tocalculate the AT-III dilution series, ratio of AT-III to FIXa, and theVi/Vo ratios for each FIXa replicate at each experimental AT-IIIconcentration. Non-linear regression analyses of residual FIXa activity(expressed as Vi/Vo) versus AT-III concentration was processed usingXLfit4 and a hyperbolic inhibition equation of the form((C+(Amp*(1−(X/(K_(0.5)+X))))); where C=the offset (fixed at 0 to permitextrapolation of data sets that did not reach 100% inhibition during thecourse of the assay), Amp=the amplitude of the fit and K_(0.5), whichcorresponds to the concentration of AT-III required for half-maximalinhibition under the assay conditions. For several FIXa variants,AT-III/LMWH inhibited less than 10-15% of the total protease activity atthe highest tested concentration of AT-III, representing an upper limitof detection for the assay under standard screening conditions. Variantswith less than 10% maximal inhibition were therefore assigned a lowerlimit K_(0.5) value of 999 nM and in most cases are expected to haveAT-III resistances much greater than the reported value.

Table 20 provides the results of the assays that were performed usingAT-III/LMWH. The results are presented both as the fitted K_(0.5)parameter and as a representation of the extent of AT-III resistance foreach variant compared to the wild-type FIXa expressed as a ratio oftheir fitted K_(0.5) values (K_(0.5) variant/K_(0.5) wild-type). Wherethe K_(0.5) parameter of the FIXa variant was compared to wild-typeFIXa, it was compared to a recombinant wild-type FIXa polypeptide thatwas expressed and purified using the same conditions as used for thevariant FIXa polypeptides to ensure that any differences in activitywere the result of the mutation(s), and not the result of differencesin, for example, post-translational modifications associated withdifferent expression systems. Thus, the wild-type FIXa polypeptide usedfor comparison was the recombinant wild-type FIXa generated from cloningthe FIX gene set forth in SEQ ID NO:1 and expressed from CHOX cells as apolypeptide with an amino acid sequence set forth in SEQ ID NO:3, asdescribed in Example 1 (i.e. Catalyst Biosciences WT FIX polypeptide).Several FIXa variants exhibited greater than 20-fold increasedresistance to AT-III compared to wild type FIXa (Catalyst Biosciences WTFIXa). For example, FIXa-R318A/R403A, FIXa-R318E/R340E, FIXa-R318A,FIXa-R318E, FIXa-K400E, FIXa-R338E/R403E and FIXa-K400A/R403A are amongthe group that exhibited significant resistance to AT-III.

TABLE 20 Inhibition of FIXa variants by AT-III/LMWH Mutation MutationK_(0.5) ±S.D. K_(0.5-mut)/ (Mature FIX Numbering) (ChymotrypsinNumbering) (nM) (nM) % CV K_(0.5-wt) n Plasma Purified FIXa PlasmaPurified FIXa 20.2 6.7 33% 0.7 3 BeneFIX (T148A) BeneFIX (T[148]A) 27.34.7 17% 0.9 2 Catalyst Biosciences WT Catalyst Biosciences WT 29.4 7.325% 1.0 10 A103N/N105S A[103]N/N[105]S 31.1 n/a n/a 1.1 1 D104N/K106SD[104]N/K[106]S 26.1 n/a n/a 0.9 1 K106N/V108S K[106]N/V[108]S 47.7 n/an/a 1.6 1 D85N D[85]N 33.1 n/a n/a 1.1 1 T148A T[148]A 22.9 1.7 8% 0.8 4D203N/F205T D39N/F41T 154.1 50.1 33% 5.2 4 I251S I86S 22.6 n/a n/a 0.8 1D85N/I251S D[85]N/I86S 28.3 n/a n/a 1.0 1 D85N/D104N/K106S/I251SD[85]N/D[104]N/K[106]S/I86S 32.1 n/a n/a 1.1 1 A262S A95bS 25.3 n/a n/a0.9 1 K413N K243N 34.2 n/a n/a 1.2 1 E410N E240N 24.8 7.8 31% 0.8 3E239N E74N 191.8 61.0 32% 6.5 3 T241N/H243S T76N/H78S 35.4 n/a n/a 1.2 1K247N/N249S K82N/N84S 23.1 n/a n/a 0.8 1 L321N L153N 39.0 n/a n/a 1.3 1F314N/H315S F145N/H147S 191.8 59.8 31% 6.5 3 S319N/L321S S151N/L153S113.4 n/a n/a 3.9 1 N260S N95S 64.6 n/a n/a 2.2 1 Y284N Y117N 36.7 n/an/a 1.2 1 R318A R150A 896.2 189.2 21% 30.5 2 R318E R150E 861.1 21.8 3%29.3 2 R318Y R150Y 395.1 6.3 2% 13.5 2 R312Q R143Q 52.7 5.1 10% 1.8 2R312A R143A 51.9 1.3 3% 1.8 2 R312Y R143Y 323.0 13.7 4% 11.0 2 R312LR143L 25.5 2.9 11% 0.9 2 V202M V38M 20.3 5.1 25% 0.7 2 V202Y V38Y 27.26.9 25% 0.9 2 D203M D39M 18.6 6.9 37% 0.6 2 D203Y D39Y 31.1 0.3 1% 1.1 2A204M A40M 45.8 11.1 24% 1.6 2 A204Y A40Y 43.4 22.3 51% 1.5 2K400A/R403A K230A/R233A 585.0 160.5 27% 19.9 2 K400E/R403E K230E/R233E299.0 206.5 69% 10.2 2 R403A R233A 164.3 88.7 54% 5.6 2 R403E R233E264.2 80.9 31% 9.0 2 K400A K230A 384.0 121.1 32% 13.1 2 K400E K230E614.8 71.4 12% 20.9 2 K293E K126E 290.2 42.1 15% 9.9 2 K293A K126A 194.138.0 20% 6.6 2 R333A R165A 225.7 72.7 32% 7.7 2 R333E R165E 345.6 1.7 0%11.8 2 R338A R170A 56.2 8.4 15% 1.9 2 R338E R170E 238.4 n/a n/a 8.1 1R338A/R403A R170A/R233A 418.5 150.9 36% 14.2 2 R338E/R403E R170E/R233E601.6 241.5 40% 20.5 2 K293A/R403A K126A/R233A 486.3 114.9 24% 16.6 2K293E/R403E K126E/R233E 342.0 4.9 1% 11.6 2 K293A/R338A/R403AK126A/R170A/R233A 497.1 85.9 17% 16.9 2 K293E/R338E/R403EK126E/R170E/R233E 418.5 150.9 36% 14.2 2 R318A/R403A R150A/R233A 999.0n/a n/a 34.0 2 R318E/R403E R150E/R233E 999.0 n/a n/a 34.0 2 A K_(0.5)value of 999 nM indicates the lower limit value for those variants withless than 10% inhibition under the conditions of the assay.

B. Inhibition of FIXa by the Antithrombin/UFH Complex

Additional experiments were performed to assess the inhibition of FIXavariants by AT-III/UFH (unfractionated full-length heparin) using thesame assay as described above with minor modifications. Full-length,unfractionated heparin (Calbiochem) was used instead of low molecularweight heparin (LMWH) to observe the effects of FIXa variant mutationson the increased rate of the inhibition reaction due to the “templating”effect provided by longer heparin chains (see e.g., Olson et al. (2004)Thromb Haemost 92(5), 929-939).

For inhibition reactions in the presence of UFH, a 70 nM, 600 nM, 2000nM, 6000 or 10000 nM solutions of AT-III/UFH (final 1 μM UFH) wereprepared by dilution of a 20 μM stock of plasma purified human AT-III(Molecular Innovations) into a solution of excess UFH (2 to 20 μM) in a1.4 mL volume of 1× Buffer A (50 mM Tris, 100 mM NaCl, 10 mM CaCl₂,0.01% Tween-20, pH 7.4). AT-III/UFH solutions were also incubated for 30minutes at room temperature before being serially diluted 1.5-fold in a96 deep-well polypropylene plate with a final volume of 460 μL 1× BufferA containing 1 μM UFH. The final dilutions of AT-III for the modifiedassay were dependent on the starting concentration of AT-III and rangedfrom 70 nM-0 nM, 600 nM-0 nM, 100 nM-0 nM or 5000 nM-0 nM (i.e. rowsA-H). Those variants, which showed increased resistance to AT-IIIinhibition under the standard conditions, were further tested usinghigher concentrations of AT-III. A total of 35 μL of each AT-IIIdilution was aliquoted into their respective rows of a 96-well V-bottomstorage plate to fill all columns (i.e. 1-12). FIXa variants wereinitially diluted to 100 nM in 1× Buffer A. Subsequently, 15 μL of each100 nM FIXa variant was diluted to a concentration of 0.6 nM in 2.0 mLof 1× Buffer A and then 70 μL of this solution was aliquoted into a96-well V-bottom storage plate according to the same predefined platemap (4 FIXa variants per plate).

Assay reactions were initiated using a BioMek FX liquid handling systemprogrammed to dispense 35 μL of the FIXa solutions into the platescontaining 35 μL of each dilution of AT-III/heparin per well for a totalof two duplicate assay plates for each FIXa variant. The finalinhibition assay conditions were: 0.3 nM FIXa and AT-III dilutionsranging from 35 nM to 0 nM, 300 nM to 0 nM, 1000 nM to 0 nM, 3000 nM to0 nM or 5000 nM to 0 nM in UFH ranging from 1 μM to 10 μM, depending ofthe highest AT-III concentration so that the heparin remained in excessInhibition reactions were further incubated for 10 seconds at roomtemperature (˜25° C.) before a 40 μL aliquot of the reaction wastransferred by the BioMek FX to a 96-well black half-area platecontaining 20 μL of 2.5 mM Mesyl-D-CHG-Gly-Arg-AMC per well in assayBuffer C (50 mM Tris, 100 mM NaCl, 10 mM CaCl₂, 0.01% Tween-20, pH 7.4,82% ethylene glycol and 5 mg/mL polybrene). Polybrene (hexadimethrinebromide) at a final concentration of 5 mg/mL was added to Buffer C toquench the AT-III/UFH reaction. Residual activity of FIXa was assessedby following the initial rates of substrate cleavage for 60 minutes in afluorescence reader set to 25° C. The final assay conditions fordetermination of residual activity were 0.2 nM FIXa variant, 0.83 mMMesyl-D-CHG-Gly-Arg-AMC, 30% ethylene glycol and 5 mg/mL polybrene in 50mM Tris, 100 mM NaCl, 10 mM CaCl₂, 0.01% Tween-20, pH 7.4. Data analyseswere performed as described above for AT-III/LMWH inhibition assays.

As found with LMWH, AT-III/UFH inhibited less than 10-15% of the of thetotal protease activity for a number of FIXa variants at the highesttested concentrations of AT-III, thus representing an upper limit ofdetection for the assay under standard screening conditions. Thesevariants with less than 10% maximal inhibition were therefore assigned alower limit K_(0.5) value of 999 nM and in most cases are expected tohave AT-III resistances much greater than the reported value. SeveralFIXa variants that were initially given a K_(0.5) value of 999 nM wereretested at higher AT-III concentrations, expanding the sensitivity ofthe assay and providing clear levels of AT-III resistance. If thesevariants still maintained less than 10% maximal inhibition at thehighest test AT-III concentrations (1000 nM to 5000 nM) a lower limitK_(0.5) value of 9999 nM was assigned, thus these variants are expectedto have AT-III resistances much greater than the reported value.

Tables 21-22 provide the results of the assays that were performed usingAT-III/UFH. Table 22 reflects data for additional FIXa variants andprovides new overall averages calculated to include additionalexperimental replicates (n) for FIXa variants in Table 21. The resultsare presented both as the fitted K_(0.5) parameter and as arepresentation of the extent of AT-III resistance for each variantcompared to the wild-type FIXa expressed as a ratio of their fittedK_(0.5) values (K_(0.5) variant/K_(0.5) wild-type). Several FIXavariants exhibited greater than 100 to 500-fold increased resistance toAT-III compared to wild-type FIXa. For example, FIXa-R318A/R403A,FIXa-R318A, FIXa-R318Y, FIXa-R338A/R403A FIXa-D203N/F205T/R318Y,FIXa-R318Y/R338E/R403E, FIXa-R318Y/R338E/R403E, FIXa-R318Y/R338E/E410N,R318Y/R338E/T343R/N346Y/R403E/E410N and FIXa-R318Y/R403E/E410N are amongthis group, which exhibited significant resistance to AT-III.

TABLE 21 Inhibition of FIXa variants by AT-III/UFH Mutation MutationK_(0.5) ±S.D. K_(0.5-mut)/ (Mature FIX Numbering) (ChymotrypsinNumbering) (nM) (nM) % CV K_(0.5-wt) n BeneFIX Benefix ® BeneFIXBenefix ® 18 8 44% 0.9 51 Coagulation FIX (T148A) Coagulation FIX(T[148]A) Plasma Purified FIXa Plasma Purified FIXa 30 4 14% 1.6 5Catalyst Biosciences WT Catalyst Biosciences WT 19 7 34% 1.0 15 N157DN[157]D 17 4 23% 0.9 2 Y155F Y[155]F 13 0 1% 0.7 2 A103N/N105S/Y155FA[103]N/N[105]S/Y[155]F 11 6 49% 0.6 2 D104N/K106S/Y155FD[104]N/K[106]S/Y[155]F 6 2 33% 0.3 2 A103N/N105S A[103]N/N[105]S 20 314% 1.0 2 D104N/K106S D[104]N/K[106]S 20 2 9% 1.0 2 K106N/V108SK[106]N/V[108]S 24 0 1% 1.2 2 D85N D[85]N 17 3 15% 0.9 4 T148A T[148]A21 8 39% 1.1 10 K5A K[5]A 22 3 15% 1.2 2 D64N D[64]N 18 0 1% 0.9 2 D64AD[64]A 16 2 12% 0.8 2 N167D N[167]D 12 2 14% 0.6 2 N167Q N[167]Q 12 1 8%0.6 2 S61A S[61]A 19 3 18% 1.0 2 S53A S[53]A 27 4 16% 1.4 2 T159AT[159]A 33 7 23% 1.7 2 T169A T[169]A 17 6 36% 0.9 2 T172A T[172]A 16 321% 0.8 2 T179A T[179]A 24 2 7% 1.2 2 Y155H Y[155]H 25 4 15% 1.3 2 Y155QY[155]Q 23 0 1% 1.2 2 S158A S[158]A 20 1 5% 1.0 2 S158D S[158]D 15 2 16%0.8 2 S158E S[158]E 14 1 10% 0.7 2 N157Q N[157]Q 16 2 11% 0.8 2D203N/F205T D39N/F41T 271 51 19% 14.0 5 D85N/D203N/F205TD[85]N/D39N/F41T 587 65 11% 30.3 2 K228N K63N 29 13 46% 1.5 6 D85N/K228ND[85]N/K63N 34 3 7% 1.7 2 A103N/N105S/K228N A[103]N/N[105]S/K63N 46 1736% 2.4 2 D104N/K106S/K228N D[104]N/K[106]S/K63N 41 21 52% 2.1 2Y155F/K228N Y[155]F/K63N 15 n.d. n.d. 0.8 1 D104N/K106S/Y155F/K228ND[104]N/K[106]S/Y[155]F/K63N 49 5 9% 2.5 2 I251S I86S 28 8 28% 1.4 4D85N/I251S D[85]N/I86S 19 6 30% 1.0 2 D85N/D104N/K106S/I251SD[85]N/D[104]N/K[106]S/I86S 28 11 41% 1.4 2 A103N/N105S/I251SA[103]N/N[105]S/I86S 42 14 33% 2.2 3 D104N/K106S/I251SD[104]N/K[106]S/I86S 32 5 16% 1.6 2 Y155F/I251S Y[155]F/I86S 18 3 19%0.9 2 A262S A95bS 25 5 21% 1.3 2 K413N K243N 27 13 48% 1.4 2 E410N E240N9 2 27% 0.5 4 E239N E74N 132 21 16% 6.8 2 T241N/H243S T76N/H78S 21 1256% 1.1 2 K247N/N249S K82N/N84S 22 4 18% 1.1 4 Y155F/K247N/N249SY[155]F/K82N/N84S 13 3 24% 0.7 4 A103N/N105S/K247N/N249SA[103]N/N[105]S/K82N/N84S 53 29 55% 2.7 4 D104N/K106S/K247N/N249SD[104]N/K[106]S/K82N/N84S 19 2 9% 1.0 2 D104N/K106S/Y155F/K247N/D[104]N/K[106]S/Y[155]F/K82N/ 27 2 9% 1.4 2 N249S N84S L321N L153N 25 625% 1.3 2 F314N/H315S F145N/H147S 104 27 26% 5.4 4 S319N/L321SS151N/L153S 65 11 17% 3.4 2 N260S N95S 312 283 91% 16.1 13D104N/K106S/N260S D[104]N/K[106]S/N95S 228 82 36% 11.8 2 Y155F/N260SY[155]F/N95S 77 16 21% 4.0 2 D104N/K106S/Y155F/N260SD[104]N/K[106]S/Y[155]F/N95S 292 37 13% 15.1 2 Y284N Y117N 41 25 63% 2.15 R318N/A320S R150N/A152S 999 0 0% 51.7 2 R318A R150A 4145 1297 31%214.3 2 R318E R150E 10000 0 0% 517.0 2 R318Y R150Y 1976 430 22% 102.2 2R312Q R143Q 33 9 26% 1.7 2 R312A R143A 31 0 1% 1.6 2 R312Y R143Y 2499350 14% 129.2 2 R312L R143L 17 1 5% 0.9 2 V202M V38M 14 2 14% 0.7 2V202Y V38Y 18 3 14% 0.9 2 D203M D39M 11 0 1% 0.6 2 D203Y D39Y 16 3 21%0.8 2 A204M A40M 29 3 9% 1.5 2 A204Y A40Y 24 1 3% 1.2 2 K400A/R403AK230A/R233A 999 0 0% 51.7 2 K400E/R403E K230E/R233E 999 0 0% 51.7 2R403A R233A 190 34 18% 9.8 4 R403E R233E 731 14 2% 37.8 2 K400A K230A114 3 3% 5.9 2 K400E K230E 301 27 9% 15.6 2 K293E K126E 187 25 13% 9.7 2K293A K126A 82 1 1% 4.2 2 R333A R165A 235 54 23% 12.1 2 R333E R165E 9990 0% 51.7 2 R338A R170A 33 3 10% 1.7 2 R338E R170E 222 124 56% 11.5 8R338A/R403A R170A/R233A 328 106 32% 17.0 6 R338E/R403E R170E/R233E 60001089 18% 310.2 2 K293A/R403A K126A/R233A 999 0 0% 51.7 2 K293E/R403EK126E/R233E 999 0 0% 51.7 2 K293A/R338A/R403A K126A/R170A/R233A 999 0 0%51.7 2 K293E/R338E/R403E K126E/R170E/R233E 999 0 0% 51.7 2 R318A/R403AR150A/R233A 999 0 0% 51.7 2 R318E/R403E R150E/R233E 999 0 0% 51.7 2R318Y/E410N R150Y/E240N 607 164 27% 31.4 4 R338E/E410N R170E/E240N 92 1415% 4.7 4 R338E/R403E/E410N R170E/R233E/E240N 2351 168 7% 121.5 2R318Y/R338E/R403E R150Y/R170E/R233E 10000 0 0% 517.0 7 D203N/F205T/K228ND39N/F41T/K63N 822 69 8% 42.5 2 D203N/F205T/E410N D39N/F41T/E240N 377 205% 19.5 2 D203N/F205T/R338E D39N/F41T/R170E 1170 180 15% 60.5 2D203N/F205T/R338A D39N/F41T/R170A 423 61 14% 21.9 2 D203N/F205T/R318YD39N/F41T/R150Y 7226 133 2% 373.6 2 D203N/F205T/R338E/R403ED39N/F41T/R170E/R233E 1520 162 11% 78.6 2 K228N/E410N K63N/E240N 36 720% 1.9 2 K228N/R338E K63N/R170E 108 8 7% 5.6 2 K228N/R338A K63N/R170A51 7 14% 2.7 2 K228N/R318Y K63N/R150Y 3414 73 2% 176.5 2K228N/R338E/R403E K63N/R170E/R233E 1679 239 14% 86.8 2 R403E/E410NR233E/E240N 279 26 9% 14.4 2 R318Y/R338E/E410N R150Y/R170E/E240N 34581033 30% 178.8 5 D104N/K106S/R318Y/R338E/ D[104]N/K[106]S/R150Y/R170E/6328 4241 67% 327.2 4 E410N E240N Y155F/R318Y/R338E/E410NY[155]F/R150Y/R170E/E240N 1098 1095 100% 56.8 7 K228N/R318Y/E410NK63N/R150Y/E240N 475 83 17% 24.6 2 R318Y/R403E/E410N R150Y/R233E/E240N7072 1387 20% 365.6 2 R318Y/R338E/R403E/E410N R150Y/R170E/R233E/E240N5881 4757 81% 304.1 4 A103N/N105S/R318Y/R338E/A[103]N/N[105]S/R150Y/R170E/ 9193 1037 11% 475.3 4 R403E/E410NR233E/E240N D104N/K106S/R318Y/R338E/ D[104]N/K[106]S/R150Y/R170E/ 100000 0% 517.0 2 R403E/E410N R233E/E240N Y155F/R318Y/R338E/R403E/Y[155]F/R150Y/R170E/R233E/ 10000 0 0% 517.0 2 E410N E240NA103N/N105S/Y155F/R318Y/ A[103]N/N[105]S/Y[155]F/R150Y/ 10000 0 0% 517.02 R338E/R403E/E410N R170E/R233E/E240N D104N/K106S/Y155F/R318Y/D[104]N/K[106]S/Y[155]F/R150Y/ 10000 0 0% 517.0 2 R338E/R403E/E410NR170E/R233E/E240N D203N/F205T/R318Y/E410N D39N/F41T/R150Y/E240N 1280 22017% 66.2 2 R333S R165S 720 67 9% 37.2 2 R338L R170L 121 6 5% 6.3 2 K316NK148N 56 2 4% 2.9 2 K316A K148A 63 15 24% 3.2 2 K316E K148E 183 2 1% 9.52 K316S K148S 77 15 19% 4.0 2 K316M K148M 9 2 24% 0.5 2 E239S E74S 10112 12% 5.2 2 E239A E74A 30 14 47% 1.6 3 E239R E74R 65 17 26% 3.3 2 E239KE74K 19 4 22% 1.0 2 H257F H92F 12 1 11% 0.6 2 H257Y H92Y 20 2 12% 1.0 2H257E H92E 25 12 48% 1.3 3 H257S H92S 23 21 89% 1.2 3 T412A T242A 25 314% 1.3 4 T412V T242V 23 4 16% 1.2 4 E410N/T412A E240N/T242A 10 1 7% 0.52 E410N/T412V E240N/T242V 11 3 24% 0.6 2 E410Q E240Q 24 14 60% 1.2 4E410S E240S 26 16 63% 1.3 7 E410A E240A 42 24 58% 2.2 6 E410D E240D 41 25% 2.1 2 N346D N178D 222 176 79% 11.5 5 Y155F/N346D Y[155]F/N178D 223102 46% 11.5 2 N346Y N178Y 36 2 7% 1.9 4 Y345A Y177A 96 87 90% 5.0 13Y345T Y177T 16 0 0% 0.8 2 T343R T175R 7 1 10% 0.4 2 T343E T175E 55 8 15%2.8 2 T343Q T175Q 13 3 25% 0.7 2 F342I F174I 98 10 11% 5.1 2 T343R/Y345TT175R/Y177T 6 0 4% 0.3 2 R318Y/R338E R150Y/R170E 397 50 12% 20.5 2Y259F/K265T/Y345T Y94F/K98T/Y177T 6 0 2% 0.3 2 K228N/I251S K63N/I86S 7316 22% 3.8 2 K228N/R318Y/R338E/R403E/ K63N/R150Y/R170E/R233E/E240N 100000 0% 517.0 2 E410N Y155F/K228N/R318Y/R338E/ Y[155]F/K63N/R150Y/R170E/10000 0 0% 517.0 2 R403E/E410N R233E/E240N D85N/K228N/R318Y/R338E/D[85]N/K63N/R150Y/R170E/ 10000 0 0% 517.0 2 R403E/E410N R233E/E240NI251S/R318Y/R338E/R403E/ I86S/R150Y/R170E/R233E/E240N 10000 0 0% 517.0 2E410N D104N/K106S/I251S/R318Y/ D[104]N/K[106]S/I86S/R150Y/ 10000 0 0%517.0 3 R338E/R403E/E410N R170E/R233E/E240N Y155F/I251S/R318Y/R338E/Y[155]F/I86S/R150Y/R170E/R233E/ 10000 0 0% 517.0 2 R403E/E410N E240NI251S/R318Y/R338E/E410N I86S/R150Y/R170E/E240N 5855 3889 66% 302.7 7D104N/K106S/I251S/R318Y/ D[104]N/K[106]S/I86S/R150Y/ 8985 1436 16% 464.52 R338E/E410N R170E/E240N F314N/K316S F145N/K148S 1221 505 41% 63.1 4K247N/N249S/R318Y/R338E/ K82N/N84S/R150Y/R170E/R233E/ 8076 2967 37%417.6 9 R403E/E410N E240N Y155F/K247N/N249S/R318Y/Y[155]F/K82N/N84S/R150Y/R170E/ 10000 0 0% 517.0 3 R338E/R403E/E410NR233E/E240N A103N/N105S/K247N/N249S/ A[103]N/N[105]S/K82N/N84S/ 2497 77231% 129.1 4 R318Y/R338E/R403E/E410N R150Y/R170E/R233E/E240ND104N/K106S/K247N/N249S/ D[104]N/K[106]S/K82N/N84S/ 10000 0 0% 517.0 2R318Y/R338E/R403E/E410N R150Y/R170E/R233E/E240N K247N/N249S/R318Y/R338E/K82N/N84S/R150Y/R170E/E240N 1514 631 42% 78.3 3 E410NY155F/K247N/N249S/R318Y/ Y[155]F/K82N/N84S/R150Y/R170E/ 3875 846 22%200.4 2 R338E/E410N E240N R318Y/R338E/R403E/E410SR150Y/R170E/R233E/E240S 10000 0 0% 517.0 2 R318Y/R338E/E410SR150Y/R170E/E240S 5402 2785 52% 279.3 5 K228N/K247N/N249S K63N/K82N/N84S85 19 22% 4.4 2 D104N/K106S/Y155F/K228N/ D[104]N/K[106]S/Y[155]F/K63N/32 12 37% 1.6 4 K247N/N249S K82N/N84S D104N/K106S/K228N/K247N/D[104]N/K[106]S/K63N/K82N/ 41 18 45% 2.1 10 N249S N84SY155F/K228N/K247N/N249S Y[155]F/K63N/K82N/N84S 27 6 22% 1.4 2K228N/K247N/N249S/R318Y/ K63N/K82N/N84S/R150Y/R170E/ 10000 0 0% 517.0 2R338E/R403E/E410N R233E/E240N R318Y/R338E/R403E/E410N/R150Y/R170E/R233E/E240N/ 10000 0 0% 517.0 2 T412V T242VR318Y/R338E/R403E/E410N/ R150Y/R170E/R233E/E240N/ 10000 0 0% 517.0 2T412A T242A R318Y/R338E/R403E/T412A R150Y/R170E/R233E/T242A 10000 0 0%517.0 2 R318Y/R338E/T412A R150Y/R170E/T242A 7661 3243 42% 396.1 9R318Y/R338E/E410N/T412V R150Y/R170E/E240N/T242V 10000 0 0% 517.0 2N260S/R318Y/R338E/R403E/ N95S/R150Y/R170E/R233E/E240N 10000 0 0% 517.0 2E410N D104N/K106S/N260S/R318Y/ D[104]N/K[106]S/N95S/R150Y/ 10000 0 0%517.0 3 R338E/R403E/E410N R170E/R233E/E240N Y155F/N260S/R318Y/R338E/Y[155]F/N95S/R150Y/R170E/ 9696 527 5% 501.3 3 R403E/E410N R233E/E240NR318Y/R338E/N346D/R403E/ R150Y/R170E/N178D/R233E/ 10000 0 0% 517.0 2E410N E240N Y155F/R318Y/R338E/N346D/ Y[155]F/R150Y/R170E/N178D/ 10000 00% 517.0 2 R403E/E410N R233E/E240N K247N/N249S/N260S K82N/N84S/N95S 15738 24% 8.1 3 Y155F/K247N/N249S/N260S Y[155]F/K82N/N84S/N95S 152 39 26%7.9 3 D[104]N/K[106]S/K247N/N249S/ D[104]N/K[106]S/K82N/N84S/ 1262 40 3%65.3 2 N260S N95S D[104]N/K[106]S/Y[155]F/K247N/D[104]N/K[106]S/Y[155]F/K82N/ 692 84 12% 35.8 2 N249S/N260S N84S/N95SK247N/N249S/N260S/R318Y/ K82N/N84S/N95S/R150Y/R170E/ 5560 3872 70% 287.53 R338E/R403E/E410N R233E/E240N Y155F/N260S/N346D Y[155]F/N95S/N178D1382 477 35% 71.4 2 R318Y/R338E/T343R/R403E/ R150Y/R170E/T175R/R233E/10000 0 0% 517.0 2 E410N E240N R338E/T343R R170E/T175R 16 6 38% 0.8 2 AK_(0.5) value of 999 nM indicates the lower limit value for thosevariants with less than 10% inhibition under the conditions of thestandard assay (35 nM-0 nM AT-III). Variants with >50% of WTk_(cat)/K_(M) (see Example 4, Table 14) and initially given a K_(0.5)value of 999 nM were retested at higher AT-III concentrations, expandingin the sensitivity of the assay. A K_(0.5) value of 9999 nM indicatesthe lower limit value for those variants with less than 10% inhibitionunder the conditions of the expanded sensitivity assay (1000 nM-0 nMAT-III and 5000-0 nM AT-III).

TABLE 22 Inhibition of FIXa variants by AT-III/UFH Mutation MutationK_(0.5) ±S.D. K_(0.5-mut)/ (Mature FIX Numbering) (ChymotrypsinNumbering) (nM) (nM) % CV K_(0.5-wt) n BeneFIX Benefix ® CoagulationBeneFIX Benefix ® 17 8 47% 0.9 55 FIX (T148A) Coagulation FIX (T[148]A)Plasma Purified FIXa Plasma Purified FIXa 30 4 14% 1.6 5 CatalystBiosciences WT Catalyst Biosciences WT 19 7 34% 1.0 15 N157D N[157]D 174 23% 0.9 2 Y155F Y[155]F 13 0 1% 0.7 2 A103N/N105S/Y155FA[103]N/N[105]S/Y[155]F 11 6 49% 0.6 2 D104N/K106S/Y155FD[104]N/K[106]S/Y[155]F 6 2 33% 0.3 2 A103N/N105S A[103]N/N[105]S 20 314% 1.0 2 D104N/K106S D[104]N/K[106]S 20 2 9% 1.0 2 K106N/V108SK[106]N/V[108]S 24 0 1% 1.2 2 D85N D[85]N 17 3 15% 0.9 4 T148A T[148]A17 10 56% 0.9 13 K5A K[5]A 22 3 15% 1.2 2 D64N D[64]N 18 0 1% 0.9 2 D64AD[64]A 16 2 12% 0.8 2 N167D N[167]D 12 2 14% 0.6 2 N167Q N[167]Q 12 1 8%0.6 2 S61A S[61]A 19 3 18% 1.0 2 S53A S[53]A 27 4 16% 1.4 2 T159AT[159]A 33 7 23% 1.7 2 T169A T[169]A 17 6 36% 0.9 2 T172A T[172]A 16 321% 0.8 2 T179A T[179]A 24 2 7% 1.2 2 Y155H Y[155]H 25 4 15% 1.3 2 Y155QY[155]Q 23 0 1% 1.2 2 S158A S[158]A 20 1 5% 1.0 2 S158D S[158]D 15 2 16%0.8 2 S158E S[158]E 14 1 10% 0.7 2 N157Q N[157]Q 16 2 11% 0.8 2D203N/F205T D39N/F41T 271 51 19% 14.0 5 D85N/D203N/F205TD[85]N/D39N/F41T 587 65 11% 30.3 2 K228N K63N 29 13 46% 1.5 6 D85N/K228ND[85]N/K63N 34 3 7% 1.7 2 A103N/N105S/K228N A[103]N/N[105]S/K63N 46 1736% 2.4 2 D104N/K106S/K228N D[104]N/K[106]S/K63N 41 21 52% 2.1 2Y155F/K228N Y[155]F/K63N 15 n.d. n.d. 0.8 1 D104N/K106S/Y155F/K228ND[104]N/K[106]S/Y[155]F/K63N 49 5 9% 2.5 2 I251S I86S 28 8 28% 1.4 4D85N/I251S D[85]N/I86S 19 6 30% 1.0 2 D85N/D104N/K106S/I251SD[85]N/D[104]N/K[106]S/I86S 28 11 41% 1.4 2 A103N/N105S/I251SA[103]N/N[105]S/I86S 42 14 33% 2.2 3 D104N/K106S/I251SD[104]N/K[106]S/I86S 32 5 16% 1.6 2 Y155F/I251S Y[155]F/I86S 18 3 19%0.9 2 A262S A95bS 25 5 21% 1.3 2 K413N K243N 27 13 48% 1.4 2 E410N E240N8 2 25% 0.4 6 E239N E74N 132 21 16% 6.8 2 T241N/H243S T76N/H78S 21 1256% 1.1 2 K247N/N249S K82N/N84S 22 4 18% 1.1 4 Y155F/K247N/N249SY[155]F/K82N/N84S 13 3 24% 0.7 4 A103N/N105S/K247N/N249SA[103]N/N[105]S/K82N/N84S 53 29 55% 2.7 4 D104N/K106S/K247N/N249SD[104]N/K[106]S/K82N/N84S 19 2 9% 1.0 2 D104N/K106S/Y155F/K247N/N249SD[104]N/K[106]S/Y[155]F/K82N/ 27 2 9% 1.4 2 N84S L321N L153N 25 6 25%1.3 2 F314N/H315S F145N/H147S 104 27 26% 5.4 4 S319N/L321S S151N/L153S65 11 17% 3.4 2 N260S N95S 312 283 91% 16.1 13 D104N/K106S/N260SD[104]N/K[106]S/N95S 228 82 36% 11.8 2 Y155F/N260S Y[155]F/N95S 77 1621% 4.0 2 D104N/K106S/Y155F/N260S D[104]N/K[106]S/Y[155]F/N95S 292 3713% 15.1 2 Y284N Y117N 41 25 63% 2.1 5 R318N/A320S R150N/A152S 999 0 0%51.7 2 R318A R150A 4145 1297 31% 214.3 2 R318E R150E 9999 0 0% 517.0 2R318Y R150Y 1976 430 22% 102.2 2 R312Q R143Q 33 9 26% 1.7 2 R312A R143A31 0 1% 1.6 2 R312Y R143Y 2499 350 14% 129.2 2 R312L R143L 17 1 5% 0.9 2V202M V38M 14 2 14% 0.7 2 V202Y V38Y 18 3 14% 0.9 2 D203M D39M 11 0 1%0.6 2 D203Y D39Y 16 3 21% 0.8 2 A204M A40M 29 3 9% 1.5 2 A204Y A40Y 24 13% 1.2 2 K400A/R403A K230A/R233A 999 0 0% 51.7 2 K400E/R403E K230E/R233E999 0 0% 51.7 2 R403A R233A 190 34 18% 9.8 4 R403E R233E 731 14 2% 37.82 K400A K230A 114 3 3% 5.9 2 K400E K230E 301 27 9% 15.6 2 K293E K126E187 25 13% 9.7 2 K293A K126A 82 1 1% 4.2 2 R333A R165A 235 54 23% 12.1 2R333E R165E 999 0 0% 51.7 2 R338A R170A 33 3 10% 1.7 2 R338E R170E 222124 56% 11.5 8 R338A/R403A R170A/R233A 328 106 32% 17.0 6 R338E/R403ER170E/R233E 6000 1089 18% 310.2 2 K293A/R403A K126A/R233A 999 0 0% 51.72 K293E/R403E K126E/R233E 999 0 0% 51.7 2 K293A/R338A/R403AK126A/R170A/R233A 999 0 0% 51.7 2 K293E/R338E/R403E K126E/R170E/R233E999 0 0% 51.7 2 R318A/R403A R150A/R233A 999 0 0% 51.7 2 R318E/R403ER150E/R233E 999 0 0% 51.7 2 R318Y/E410N R150Y/E240N 607 164 27% 31.4 4R338E/E410N R170E/E240N 92 14 15% 4.7 4 R338E/R403E/E410NR170E/R233E/E240N 2351 168 7% 121.5 2 R318Y/R338E/R403ER150Y/R170E/R233E 10000 0 0% 517.0 7 D203N/F205T/K228N D39N/F41T/K63N822 69 8% 42.5 2 D203N/F205T/E410N D39N/F41T/E240N 377 20 5% 19.5 2D203N/F205T/R338E D39N/F41T/R170E 1170 180 15% 60.5 2 D203N/F205T/R338AD39N/F41T/R170A 423 61 14% 21.9 2 D203N/F205T/R318Y D39N/F41T/R150Y 7226133 2% 373.6 2 D203N/F205T/R338E/R403E D39N/F41T/R170E/R233E 1520 16211% 78.6 2 K228N/E410N K63N/E240N 36 7 20% 1.9 2 K228N/R338E K63N/R170E108 8 7% 5.6 2 K228N/R338A K63N/R170A 51 7 14% 2.7 2 K228N/R318YK63N/R150Y 3414 73 2% 176.5 2 K228N/R338E/R403E K63N/R170E/R233E 1679239 14% 86.8 2 R403E/E410N R233E/E240N 279 26 9% 14.4 2R318Y/R338E/E410N R150Y/R170E/E240N 3458 1033 30% 178.8 5D104N/K106S/R318Y/R338E/E410N D[104]N/K[106]S/R150Y/R170E/ 6328 4241 67%327.2 4 E240N Y155F/R318Y/R338E/E410N Y[155]F/R150Y/R170E/E240N 10981095 100% 56.8 7 K228N/R318Y/E410N K63N/R150Y/E240N 475 83 17% 24.6 2R318Y/R403E/E410N R150Y/R233E/E240N 7072 1387 20% 365.6 2R318Y/R338E/R403E/E410N R150Y/R170E/R233E/E240N 5881 4757 81% 304.1 4A103N/N105S/R318Y/R338E/R403E/ A[103]N/N[105]S/R150Y/R170E/ 9193 103711% 475.3 4 E410N R233E/E240N D104N/K106S/R318Y/R338E/R403E/D[104]N/K[106]S/R150Y/R170E/ 10000 0 0% 517.0 2 E410N R233E/E240NY155F/R318Y/R338E/R403E/E410N Y[155]F/R150Y/R170E/R233E/ 10000 0 0%517.0 2 E240N A103N/N105S/Y155F/R318Y/R338E/A[103]N/N[105]S/Y[155]F/R150Y/ 10000 0 0% 517.0 2 R403E/E410NR170E/R233E/E240N D104N/K106S/Y155F/R318Y/R338E/D[104]N/K[106]S/Y[155]F/R150Y/ 10000 0 0% 517.0 2 R403E/E410NR170E/R233E/E240N D203N/F205T/R318Y/E410N D39N/F41T/R150Y/E240N 1280 22017% 66.2 2 R333S R165S 720 67 9% 37.2 2 R338L R170L 121 6 5% 6.3 2 K316NK148N 56 2 4% 2.9 2 K316A K148A 63 15 24% 3.2 2 K316E K148E 183 2 1% 9.52 K316S K148S 77 15 19% 4.0 2 K316M K148M 9 2 24% 0.5 2 E239S E74S 10112 12% 5.2 2 E239A E74A 30 14 47% 1.6 3 E239R E74R 65 17 26% 3.3 2 E239KE74K 19 4 22% 1.0 2 H257F H92F 12 1 11% 0.6 2 H257Y H92Y 20 2 12% 1.0 2H257E H92E 25 12 48% 1.3 3 H257S H92S 23 21 89% 1.2 3 T412A T242A 25 314% 1.3 4 T412V T242V 23 4 16% 1.2 4 E410N/T412A E240N/T242A 10 1 7% 0.52 E410N/T412V E240N/T242V 11 3 24% 0.6 2 E410Q E240Q 24 14 60% 1.2 4E410S E240S 26 16 63% 1.3 7 E410A E240A 42 24 58% 2.2 6 E410D E240D 41 25% 2.1 2 N346D N178D 222 176 79% 11.5 5 Y155F/N346D Y[155]F/N178D 223102 46% 11.5 2 N346Y N178Y 36 2 7% 1.9 4 Y345A Y177A 96 87 90% 5.0 13Y345T Y177T 16 0 0% 0.8 2 T343R T175R 7 1 10% 0.4 2 T343E T175E 55 8 15%2.8 2 T343Q T175Q 13 3 25% 0.7 2 F342I F174I 98 10 11% 5.1 2 T343R/Y345TT175R/Y177T 6 0 4% 0.3 2 R318Y/R338E R150Y/R170E 397 50 12% 20.5 2Y259F/K265T/Y345T Y94F/K98T/Y177T 6 0 2% 0.3 2 K228N/I251S K63N/I86S 7316 22% 3.8 2 K228N/R318Y/R338E/R403E/E410N K63N/R150Y/R170E/R233E/ 100000 0% 517.0 2 E240N Y155F/K228N/R318Y/R338E/R403E/Y[155]F/K63N/R150Y/R170E/ 10000 0 0% 517.0 2 E410N R233E/E240ND85N/K228N/R318Y/R338E/R403E/ D[85]N/K63N/R150Y/R170E/ 10000 0 0% 517.02 E410N R233E/E240N I251S/R318Y/R338E/R403E/E410NI86S/R150Y/R170E/R233E/E240N 10000 0 0% 517.0 2D104N/K106S/I251S/R318Y/R338E/ D[104]N/K[106]S/I86S/R150Y/ 10000 0 0%517.0 3 R403E/E410N R170E/R233E/E240N Y155F/I251S/R318Y/R338E/R403E/D[104]N/K[106]S/I86S/R150Y/ 10000 0 0% 517.0 2 E410N R170E/R233E/E240NI251S/R318Y/R338E/E410N I86S/R150Y/R170E/E240N 5855 3889 66% 302.7 7D104N/K106S/I251S/R318Y/R338E/ D[104]N/K[106]S/I86S/R150Y/ 8985 1436 16%464.5 2 E410N R170E/E240N F314N/K316S F145N/K148S 1221 505 41% 63.1 4K247N/N249S/R318Y/R338E/R403E/ K82N/N84S/R150Y/R170E/R233E/ 8076 296737% 417.6 9 E410N E240N Y155F/K247N/N249S/R318Y/R338E/Y[155]F/K82N/N84S/R150Y/ 10000 0 0% 517.0 3 R403E/E410NR170E/R233E/E240N A103N/N105S/K247N/N249S/R318Y/A[103]N/N[105]S/K82N/N84S/ 2497 772 31% 129.1 4 R338E/R403E/E410NR150Y/R170E/R233E/E240N D104N/K106S/K247N/N249S/R318Y/D[104]N/K[106]S/K82N/N84S/ 10000 0 0% 517.0 2 R338E/R403E/E410NR150Y/R170E/R233E/E240N K247N/N249S/R318Y/R338E/E410NK82N/N84S/R150Y/R170E/E240N 1514 631 42% 78.3 3Y155F/K247N/N249S/R318Y/R338E/ Y[155]F/K82N/N84S/R150Y/ 3875 846 22%200.4 2 E410N R170E/E240N R318Y/R338E/R403E/E410SR150Y/R170E/R233E/E240S 10000 0 0% 517.0 2 R318Y/R338E/E410SR150Y/R170E/E240S 5402 2785 52% 279.3 5 K228N/K247N/N249S K63N/K82N/N84S85 19 22% 4.4 2 D104N/K106S/Y155F/K228N/K247N/D[104]N/K[106]S/Y[155]F/K63N/ 32 12 37% 1.6 4 N249S K82N/N84SD104N/K106S/K228N/K247N/ D[104]N/K[106]S/K63N/K82N/ 41 18 45% 2.1 10N249S N84S Y155F/K228N/K247N/N249S Y[155]F/K63N/K82N/N84S 27 6 22% 1.4 2K228N/K247N/N249S/R318Y/R338E/ K63N/K82N/N84S/R150Y/R170E/ 10000 0 0%517.0 2 R403E/E410N R233E/E240N R318Y/R338E/R403E/E410N/T412VR150Y/R170E/R233E/E240N/ 10000 0 0% 517.0 2 T242VR318Y/R338E/R403E/E410N/T412A R150Y/R170E/R233E/E240N/ 10000 0 0% 517.02 T242A R318Y/R338E/R403E/T412A R150Y/R170E/R233E/T242A 10000 0 0% 517.02 R318Y/R338E/T412A R150Y/R170E/T242A 7661 3243 42% 396.1 9R318Y/R338E/E410N/T412V R150Y/R170E/E240N/T242V 4871 4173 86% 251.8 9N260S/R318Y/R338E/R403E/E410N N95S/R150Y/R170E/R233E/ 10000 0 0% 517.0 2E240N D104N/K106S/N260S/R318Y/R338E/ D[104]N/K[106]S/N95S/R150Y/ 10000 00% 517.0 3 R403E/E410N R170E/R233E/E240N Y155F/N260S/R318Y/R338E/R403E/Y[155]F/N95S/R150Y/R170E/ 9696 527 5% 501.3 3 E410N R233E/E240NR318Y/R338E/N346D/R403E/E410N R150Y/R170E/N178D/R233E/ 10000 0 0% 517.02 E240N Y155F/R318Y/R338E/N346D/R403E/ Y[155]F/R150Y/R170E/N178D/ 100000 0% 517.0 2 E410N R233E/E240N K247N/N249S/N260S K82N/N84S/N95S 157 3824% 8.1 3 Y155F/K247N/N249S/N260S Y[155]F/K82N/N84S/N95S 152 39 26% 7.93 D104N/K106S/K247N/N249S/N260S D[104]N/K[106]S/K82N/N84S/ 1262 40 3%65.3 2 N95S D104N/K106S/Y155F/K247N/N249S/ D[104]N/K[106]S/Y[155]F/K82N/692 84 12% 35.8 2 N260S N84S/N95S K247N/N249S/N260S/R318Y/R338E/K82N/N84S/N95S/R150Y/R170E/ 5560 3872 70% 287.5 3 R403E/E410NR233E/E240N Y155F/N260S/N346D Y[155]F/N95S/N178D 1382 477 35% 71.4 2R318Y/R338E/T343R/R403E/E410N R150Y/R170E/T175R/R233E/ 10000 0 0% 517.04 E240N R338E/T343R R170E/T175R 12 6 46% 0.6 4 T343R/N346Y T175R/N178Y 31 32% 0.1 4 R318Y/R338E/N346Y/R403E/E410N R150Y/R170E/N178Y/R233E/ 100000 0% 517.0 2 E240N R318Y/R338E/T343R/N346Y/R403E/R150Y/R170E/T175R/N178Y/ 10000 0 0% 517.0 2 E410N R233E/E240NT343R/N346D T175R/N178D 22 4 18% 1.1 2 R318Y/R338E/T343R/N346D/R403E/R150Y/R170E/T175R/N178D/ 10000 0 0% 517.0 2 E410N R233E/E240NR318Y/R338E/Y345A/R403E/E410N R150Y/R170E/Y177A/R233E/ 10000 0 0% 517.02 E240N R318Y/R338E/Y345A/N346D/R403E/ R150Y/R170E/Y177A/N178D/ 10000 00% 517.0 2 E410N R233E/E240N A K_(0.5) value of 999 nM indicates thelower limit value for those variants with less than 10% inhibition underthe conditions of the standard assay (35 nM-0 nM AT-III). Variantswith >50% of WT k_(cat)/K_(M) (see Example 4, Table 14) and initiallygiven a K_(0.5) value of 999 nM were retested at higher AT-IIIconcentrations, expanding in the sensitivity of the assay. A K_(0.5)value of 10000 nM indicates the lower limit value for those variantswith less than 10% inhibition under the conditions of the expandedsensitivity assay (1000 nM-0 nM AT-III and 5000-0 nM AT-III).C. Determination of the Second-Order Rate Constant (k_(app)) forInhibition of FIXa by the Antithrombin/UFH Complex

Additional experiments were performed to measure the second-order rateconstant for inhibition (k_(app)) of FIXa variants by AT-III/UFH usingthe same assay as described above in Example 5B with minormodifications. This method is more amenable to evaluating thesecond-order rate constants for multiple variants concurrently than thetraditional competitive kinetic or discontinuous methods (see e.g.,Olson et al. (2004) Thromb Haemost 92(5), 929-939).

For inhibition reactions in the presence of UFH, a 1000 nM solution ofAT-III/UFH were prepared by dilution of a 20 μM stock of plasma purifiedhuman AT-III (Molecular Innovations) into a solution of excess UFH (2μM) in a 1.0 mL volume of 1× Buffer A (50 mM Tris, 100 mM NaCl, 10 mMCaCl₂, 0.01% Tween-20, pH 7.4). AT-III/UFH solutions were incubated for30 minutes at room temperature prior to being serially diluted 2.0-foldin a 96 deep-well polypropylene plate with a final volume of 500 μL 1×Buffer A containing 2 μM UFH. The final dilutions of AT-III for themodified k_(app) assay ranged from 500 nM-0 nM (i.e. rows A-H). A totalof 35 μL of each AT-III dilution was aliquoted into their respectiverows of a 96-well V-bottom storage plate to fill all columns (i.e.1-12). FIXa variants were initially diluted to 100 nM in 1× Buffer A.Subsequently, 50 μL of each 100 nM FIXa variant was diluted to aconcentration of 2.0 nM in 2.5 mL of 1× Buffer A and then 70 μL of thissolution was aliquoted into a 96-well V-bottom storage plate accordingto the same predefined plate map as above (4 FIXa variants per plate).

Assay reactions were initiated using a BioMek FX liquid handling systemprogrammed to dispense 35 μL of the FIXa solutions into the platescontaining 35 μL of each dilution of AT-III/UFH per well for a total oftwo duplicate assay plates for each FIXa variant. The final inhibitionassay conditions were: 1.0 nM FIXa and AT-III dilutions ranging from 500nM to 0 nM in 1 μM UFH so that the heparin remained in excess Inhibitionreactions were further incubated for various times at room temperature(˜25° C.) depending on the expected inhibition rate constant andadjusted so that >90% inhibition could be reached at the highestconcentration of AT-III in the assay (500 nM). Typical incubation timeswere determined specifically for each variant, or class of variants, butgenerally followed the incubation times outlined in Table 23.

TABLE 23 Assay Incubation Times Based on Expected k_(app) ValuesExpected k_(app) (M⁻¹s⁻¹) FIXa/ATIII Incubation (sec) 1.0E−07 10 1.0E−0630 1.0E−05 120 1.0E−04 600 1.0E−03 3600 1.0E−02 7200

Following the desired incubation time a 40 μL aliquot of the reactionwas transferred by the BioMek FX to a 96-well black half-area platecontaining 20 μL of 2.5 mM Mesyl-D-CHG-Gly-Arg-AMC per well in assayBuffer C (50 mM Tris, 100 mM NaCl, 10 mM CaCl₂, 0.01% Tween-20, pH 7.4,82% ethylene glycol and 5 mg/mL polybrene). Polybrene (hexadimethrinebromide) at a final concentration of 5 mg/mL was added to Buffer C toquench the AT-III/UFH reaction. Residual activity of FIXa was assessedby following the initial rates of substrate cleavage for 60 minutes in afluorescence reader set to 25° C. The final assay conditions fordetermination of residual activity were 0.67 nM FIXa variant, 0.83 mMMesyl-D-CHG-Gly-Arg-AMC, 30% ethylene glycol and 5 mg/mL polybrene in 50mM Tris, 100 mM NaCl, 10 mM CaCl₂, 0.01% Tween-20, pH 7.4. Data analysesto calculate the K_(0.5) value were performed in a similar manner asthat described above for AT-III/UFH inhibition assays in Example B usingthe ActivityBase software package and the XE Runner data analysis module(IDBS Software). Using the assay set-up outlined in Example 5B underpsuedo-1st-order conditions and testing various incubation times it isthus possible to calculate the apparent second-order rate constant forinhibition by AT-III (k_(app)) using the following equations:

$\begin{matrix}{k_{app} = \frac{k_{obs}}{\left( \frac{{AT}\text{-}{III}}{S.I.} \right)}} & {{Equation}\mspace{14mu} (1)} \\{k_{obs} = \frac{\ln (2)}{t_{1/2}}} & {{Equation}\mspace{14mu} (2)}\end{matrix}$

Given that the fit value for K_(0.5)=[AT-III] at t_(1/2) (defined by thetime of the assay) all the necessary values are available to calculatek_(obs) and thus the k_(app) for inhibition of a given FIXa variant byAT-III. The calculated k_(app) value does not take into account anypotential effects of changes in the stoichiometry of inhibition (S.I.),which is given a constant value of 1.2 in the present calculations asthis value reflects what is typically reported in the literature (seee.g., Olson et al. (2004) Thromb Haemost 92(5), 929-939).

Table 24 provides the results of the second-order rate assays that wereperformed using AT-III/UFH. The results are presented both as the fittedk_(app) parameter and as a representation of the extent of AT-IIIresistance for each variant compared to the wild-type FIXa expressed asa ratio of their fitted k_(app) values (k_(app) wild-type/k_(app)variant). Several FIXa variants exhibited greater than 10,000-20,000fold increased resistance to AT-III compared to wild-type FIXa. Forexample, FIXa-R318A, FIXa-R318Y, FIXa-R338A/R403A,FIXa-R318Y/R338E/R403E, FIXa-R318Y/R338E/R403E,FIXa-K247N/N249S/R318Y/R338E/R403E, FIXa-R318Y/R338E/R403E,FIXa-K228N/I251S/R318Y/R338E/R403E/E410N, FIXa-R318Y/R338E/E410N andFIXa-R318Y/R338E/R403E/E410N are among this group, which exhibitedsignificant resistance to AT-III.

TABLE 24 Second-Order Rate Constant for Inhibition by AT-III/UFHMutation Mutation (Chymotrypsin k_(app) ±S.D. k_(app-wt)/ (Mature FIXNumbering) Numbering) (M⁻¹s⁻¹) (M⁻¹s⁻¹) % CV k_(app-mut) n BeneFIXBenefix ® BeneFIX Benefix ® 1.6E+07 1.7E+07 105% 1 8 Coagulation FIX(T148A) Coagulation FIX (T[148]A) Catalyst Biosciences WT CatalystBiosciences WT 2.4E+07 8.0E+06 33% 1 4 T148A T[148]A 1.6E+07 1.1E+07 69%1 4 D203N/F205T D39N/F41T 8.1E+05 5.3E+05 66% 30 3 D85N/D203N/F205TD[85]N/D39N/F41T 2.7E+06 4.5E+05 17% 9 2 N260S N95S 1.1E+06 2.1E+04 2%21 2 D104N/K106S/N260S D[104]N/K[106]S/N95S 7.0E+06 1.9E+06 27% 3 3R318A R150A 6.9E+05 5.6E+04 8% 35 2 R318E R150E 1.6E+04 1.2E+03 7% 1,4522 R318Y R150Y 6.4E+05 3.5E+05 55% 37 5 R312Y R143Y 2.3E+05 4.5E+04 19%102 3 R403A R233A 1.4E+06 3.1E+05 23% 18 2 R403E R233E 1.1E+05 2.4E+0421% 209 2 K400E K230E 4.1E+05 3.3E+04 8% 58 2 K293E K126E 1.2E+068.4E+04 7% 20 2 R338E R170E 2.7E+05 1.7E+05 64% 88 3 R338A/R403AR170A/R233A 8.4E+05 4.6E+04 5% 28 2 R338E/R403E R170E/R233E 6.8E+041.9E+04 28% 353 2 K293A/R403A K126A/R233A 8.1E+04 1.5E+04 18% 294 2K293A/R338A/R403A K126A/R170A/R233A 4.7E+04 7.9E+03 17% 511 2K293E/R338E/R403E K126E/R170E/R233E 3.1E+04 6.3E+03 20% 768 2R318A/R403A R150A/R233A 1.7E+04 4.7E+03 27% 1,390 2 R318Y/E410NR150Y/E240N 1.1E+06 7.9E+03 1% 22 2 R338E/E410N R170E/E240N 6.3E+067.4E+06 117% 4 10 R338E/R403E/E410N R170E/R233E/E240N 1.3E+05 1.5E+05115% 180 14 Y155F/R338E/R403E/E410N Y[155]F/R170E/R233E/E240N 3.2E+041.7E+03 5% 755 2 R318Y/R338E/R403E R150Y/R170E/R233E 1.2E+03 9.9E+02 80%19,396 7 Y155F/R318Y/R338E/R403E Y[155]F/R150Y/R170E/R233E 1.0E+035.4E+01 5% 23,242 2 D203N/F205T/K228N D39N/F41T/K63N 1.1E+06 3.7E+05 33%21 2 D203N/F205T/E410N D39N/F41T/E240N 2.0E+06 2.1E+05 10% 12 2D203N/F205T/R338E D39N/F41T/R170E 3.6E+05 2.8E+04 8% 66 2D203N/F205T/R338A D39N/F41T/R170A 8.6E+05 1.6E+05 18% 28 2D203N/F205T/R318Y D39N/F41T/R150Y 6.1E+04 2.0E+04 33% 391 2D203N/F205T/R338E/R403E D39N/F41T/R170E/R233E 2.0E+03 n.d. n.d. 12,250 1K228N/R318Y K63N/R150Y 1.2E+06 2.1E+05 17% 19 2 K228N/R338E/R403EK63N/R170E/R233E 4.2E+04 1.3E+04 31% 567 2 R403E/E410N R233E/E240N4.8E+06 2.5E+06 53% 5 5 R318Y/R338E/E410N R150Y/R170E/E240N 2.8E+052.4E+05 85% 84 8 D104N/K106S/R318Y/R338E/ D[104]N/K[106]S/R150Y/R170E/2.1E+05 4.2E+04 20% 113 2 E410N E240N Y155F/R318Y/R338E/E410NY[155]F/R150Y/R170E/E240N 4.5E+05 6.9E+04 15% 53 2 K228N/R318Y/E410NK63N/R150Y/E240N 1.9E+06 n.d. n.d. 12 1 R318Y/R403E/E410NR150Y/R233E/E240N 2.8E+04 1.8E+04 63% 856 6 Y155F/R318Y/R403E/E410NY[155]F/R150Y/R233E/E240N 8.1E+03 1.4E+02 2% 2,963 2R318Y/R338E/R403E/E410N R150Y/R170E/R233E/E240N 3.2E+03 2.0E+03 63%7,385 6 A103N/N105S/R318Y/R338E/ A[103]N/N[105]S/R150Y/R170E/ 2.6E+031.7E+02 7% 9,060 2 R403E/E410N R233E/E240N D104N/K106S/R318Y/R338E/D[104]N/K[106]S/R150Y/R170E/ 3.9E+03 1.6E+01 0% 6,154 2 R403E/E410NR233E/E240N Y155F/R318Y/R338E/R403E/ Y[155]F/R150Y/R170E/R233E/ 3.2E+038.1E+02 25% 7,464 3 E410N E240N A103N/N105S/Y155F/R318Y/A[103]N/N[105]S/Y[155]F/ 3.2E+03 6.7E+00 0% 7,531 2 R338E/R403E/E410NR150Y/R170E/R233E/E240N D104N/K106S/Y155F/R318Y/D[104]N/K[106]S/Y[155]F/ 2.9E+03 1.8E+02 6% 8,147 2 R338E/R403E/E410NR150Y/R170E/R233E/E240N D203N/F205T/R318Y/E410N D39N/F41T/R150Y/E240N5.3E+04 5.8E+03 11% 454 3 N346D N178D 3.4E+06 1.6E+06 48% 7 4Y155F/N346D Y[155]F/N178D 4.0E+06 5.4E+05 13% 6 2 N346Y N178Y 8.4E+05n.d. n.d. 28 1 Y345T Y177T 1.8E+06 7.8E+03 0% 13 2 T343R T175R 4.2E+061.0E+04 0% 6 2 T343Q T175Q 2.1E+06 5.4E+05 25% 11 2 T343R/Y345TT175R/Y177T 5.0E+06 1.8E+05 4% 5 2 R318Y/R338E R150Y/R170E 6.2E+055.4E+04 9% 39 2 K228N/R318Y/R338E/R403E/ K63N/R150Y/R170E/R233E/ 2.9E+032.2E+02 7% 8,212 2 E410N E240N Y155F/K228N/R318Y/R338E/Y[155]F/K63N/R150Y/R170E/ 4.6E+03 6.1E+02 13% 5,161 2 R403E/E410NR233E/E240N D85N/K228N/R318Y/R338E/ D[85]N/K63N/R150Y/R170E/ 3.0E+033.2E+02 11% 7,932 2 R403E/E410N R233E/E240N I251S/R318Y/R338E/R403E/I86S/R150Y/R170E/R233E/ 3.0E+03 3.5E+02 12% 7,940 2 E410N E240ND104N/K106S/I251S/R318Y/ D[104]N/K[106]S/I86S/R150Y/ 5.7E+03 8.4E+02 15%4,225 2 R338E/R403E/E410N R170E/R233E/E240N Y155F/I251S/R318Y/R338E/D[104]N/K[106]S/I86S/R150Y/ 3.3E+03 1.4E+02 4% 7,306 2 R403E/E410NR170E/R233E/E240N I251S/R318Y/R338E/E410N I86S/R150Y/R170E/E240N 2.4E+052.1E+05 89% 100 6 D104N/K106S/I251S/R318Y/ D[104]N/K[106]S/I86S/R150Y/3.2E+03 4.5E+02 14% 7,567 2 R338E/E410N R170E/E240NK247N/N249S/R318Y/R338E/ K82N/N84S/R150Y/R170E/ 2.0E+03 1.0E+03 53%12,122 2 R403E/E410N R233E/E240N Y155F/K247N/N249S/R318Y/Y[155]F/K82N/N84S/R150Y/ 1.6E+03 5.9E+02 37% 15,058 4 R338E/R403E/E410NR170E/R233E/E240N A103N/N105S/K247N/N249S/ A[103]N/N[105]S/K82N/N84S/1.7E+03 2.4E+02 14% 14,063 3 R318Y/R338E/R403E/E410N R150Y/R170E/R233E/E240N D104N/K106S/K247N/N249S/ D[104]N/K[106]S/K82N/N84S/ 3.1E+037.6E+02 24% 7,646 3 R318Y/R338E/R403E/E410N R150Y/R170E/R233E/ E240ND104N/K106S/Y155F/K247N/ D[104]N/K[106]S/Y[155]F/ 1.0E+03 2.8E+02 28%23,776 6 N249S/R318Y/R338E/R403E/ K82N/N84S/R150Y/R170E/ E410NR233E/E240N K247N/N249S/R318Y/R338E/ K82N/N84S/R150Y/R170E/ 8.6E+051.2E+05 14% 28 2 E410N E240N Y155F/K247N/N249S/R318Y/Y[155]F/K82N/N84S/R150Y/ 1.8E+05 2.2E+04 13% 136 2 R338E/E410NR170E/E240N R318Y/R338E/R403E/E410S R150Y/R170E/R233E/E240S 1.6E+031.1E+03 64% 14,483 7 R318Y/R338E/E410S R150Y/R170E/E240S 7.2E+05 4.8E+0566% 33 2 K228N/K247N/N249S/R318Y/ K63N/K82N/N84S/R150Y/ 1.1E+03 4.5E+0241% 21,766 12 R338E/R403E/E410N R170E/R233E/E240ND104N/K106S/K228N/K247N/ D[104]N/K[106]S/K63N/K82N/ 6.8E+02 3.3E+02 48%35,018 4 N249S/R318Y/R338E/R403E/ N84S/R150Y/R170E/R233E/ E410N E240NY155F/K228N/K247N/N249S/ Y[155]F/K63N/K82N/N84S/ 1.1E+03 3.9E+01 4%21,856 4 R318Y/R338E/R403E/E410N R150Y/R170E/R233E/E240NR318Y/R338E/R403E/E410N/ R150Y/R170E/R233E/E240N/ 2.9E+03 5.4E+02 19%8,296 5 T412V T242V R318Y/R338E/R403E/E410N/ R150Y/R170E/R233E/E240N/3.8E+03 1.2E+03 31% 6,322 5 T412A T242A R318Y/R338E/R403E/T412AR150Y/R170E/R233E/T242A 1.6E+03 3.8E+02 23% 14,529 2 R318Y/R338E/T412AR150Y/R170E/T242A 3.5E+05 7.2E+04 21% 69 3 R318Y/R338E/E410N/T412VR150Y/R170E/E240N/T242V 3.9E+05 2.6E+04 7% 61 2 N260S/R318Y/R338E/R403E/N95S/R150Y/R170E/R233E/ 4.4E+03 8.5E+02 19% 5,407 2 E410N E240ND104N/K106S/N260S/R318Y/ D[104]N/K[106]S/N95S/R150Y/ 2.1E+03 3.9E+02 18%11,173 2 R338E/R403E/E410N R170E/R233E/E240N Y155F/N260S/R318Y/R338E/Y[155]F/N95S/R150Y/R170E/ 2.1E+03 2.4E+02 11% 11,456 2 R403E/E410NR233E/E240N R318Y/R338E/N346D/R403E/ R150Y/R170E/N178D/R233E/ 1.1E+035.5E+02 49% 21,504 6 E410N E240N Y155F/R318Y/R338E/N346D/Y[155]F/R150Y/R170E/N178D/ 1.6E+03 6.6E+02 41% 14,831 3 R403E/E410NR233E/E240N D104N/K106S/K247N/N249S/ D[104]N/K[106]S/K82N/N84S/ 1.7E+068.7E+04 5% 14 2 N260S N95S D104N/K106S/Y155F/K247N/D[104]N/K[106]S/Y[155]F/ 3.2E+06 2.1E+05 6% 7 2 N249S/N260SK82N/N84S/N95S K247N/N249S/N260S/R318Y/ K82N/N84S/N95S/R150Y/ 1.3E+033.8E+02 30% 18,567 2 R338E/R403E/E410N R170E/R233E/E240NY155F/K247N/N249S/N260S/ Y[155]F/K82N/N84S/N95S/ 4.3E+02 3.8E+00 1%55,342 4 R318Y/R338E/R403E/E410N R150Y/R170E/R233E/E240NR318Y/R338E/T343R/R403E/ R150Y/R170E/T175R/R233E/ 3.2E+04 2.2E+04 69%749 6 E410N E240N Y155F/R318Y/R338E/T343R/ Y[155]F/R150Y/R170E/T175R/8.6E+03 5.4E+03 63% 2,774 6 R403E/E410N R233E/E240ND104N/K106S/R318Y/R338E/ D[104]N/K[106]S/R150Y/R170E/ 9.1E+03 2.4E+0327% 2,636 4 T343R/R403E/E410N T175R/R233E/E240N R338E/T343R R170E/T175R3.4E+06 4.8E+05 14% 7 2 T343R/N346Y T175R/N178Y 4.2E+06 4.0E+06 95% 6 4R318Y/R338E/N346Y/R403E/ R150Y/R170E/N178Y/R233E/ 2.8E+03 4.4E+02 16%8,498 2 E410N E240N R318Y/R338E/T343R/N346Y/ R150Y/R170E/T175R/N178Y/1.1E+04 4.3E+03 37% 2,086 4 R403E/E410N R233E/E240N T343R/N346DT175R/N178D 1.3E+06 2.3E+05 18% 18 2 R318Y/R338E/T343R/N346D/R150Y/R170E/T175R/N178D/ 5.1E+03 3.7E+01 1% 4,726 2 R403E/E410NR233E/E240N R318Y/R338E/Y345A/R403E/ R150Y/R170E/Y177A/R233E/ 7.9E+031.2E+03 16% 3,015 2 E410N E240N Y155F/K247N/N249S/R318Y/Y[155]F/K82N/N84S/R150Y/ 8.1E+02 1.6E+02 20% 29,512 4 R338E/R403ER170E/R233E K247N/N249S/R318Y/R338E/ K82N/N84S/R150Y/R170E/ 3.1E+022.1E+02 67% 76,373 4 R403E R233E Y155F/K247N/N249S/R318Y/Y[155]F/K82N/N84S/R150Y/ 7.3E+03 2.0E+01 0% 3,291 2 R403E/E410NR233E/E240N K247N/N249S/R318Y/R403E/ K82N/N84S/R150Y/R233E/ 2.7E+039.3E+02 35% 8,942 6 E410N E240N Y155F/K247N/N249S/R338E/Y[155]F/K82N/N84S/R170E/ 4.2E+04 4.3E+02 1% 572 2 R403E/E410NR233E/E240N K247N/N249S/R338E/R403E/ K82N/N84S/R170E/R233E/ 2.1E+041.5E+03 7% 1,148 2 E410N E240N R318Y/R338E/T343R/R403ER150Y/R170E/T175R/R233E 5.8E+03 8.6E+02 15% 4,118 2Y155F/R318Y/R338E/T343R/ Y[155]F/R150Y/R170E/T175R/ 2.8E+03 3.8E+02 14%8,515 6 R403E R233E R318Y/R338E/T343R/E410N R150Y/R170E/T175R/E240N5.4E+05 3.2E+05 58% 44 8 Y155F/R318Y/R338E/T343R/Y[155]F/R150Y/R170E/T175R/ 7.8E+05 6.1E+05 79% 31 4 E410N E240NR318Y/T343R/R403E/E410N R150Y/T175R/R233E/E240N 9.3E+04 1.2E+04 13% 2572 Y155F/R318Y/T343R/R403E/ Y[155]F/R150Y/T175R/R233E/ 5.5E+04 7.8E+0314% 436 4 E410N E240N R338E/T343R/R403E/E410N R170E/T175R/R233E/E240N3.4E+05 2.7E+03 1% 70 2 Y155F/R338E/T343R/R403E/Y[155]F/R170E/T175R/R233E/ 2.8E+05 1.7E+04 6% 85 4 E410N E240NY155F/K247N/N249S/R318Y/ Y[155]F/K82N/N84S/R150Y/ 8.7E+03 1.9E+03 22%2,733 8 R338E/T343R/R403E/E410N R170E/T175R/R233E/E240NK247N/N249S/R318Y/R338E/ K82N/N84S/R150Y/R170E/ 9.6E+03 2.4E+03 25%2,499 4 T343R/R403E/E410N T175R/R233E/E240N K228N/I251S/R318Y/R338E/K63N/I86S/R150Y/R170E/ 9.0E+02 2.2E+02 25% 26,598 4 R403E/E410NR233E/E240N Y155F/K228N/I251S/R318Y/ Y[155]F/K63N/I86S/R150Y/ 1.3E+032.8E+02 21% 17,778 6 R338E/R403E/E410N R170E/R233E/E240NN260S/R318Y/R338E/T343R/ N95S/R150Y/R170E/T175R/ 2.6E+03 5.6E+02 22%9,317 4 R403E/E410N R233E/E240N Y155F/N260S/R318Y/R338E/Y[155]F/N95S/R150Y/R170E/ 2.6E+03 6.6E+02 25% 9,148 4 T343R/R403E/E410NT175R/R233E/E240N K228N/K247N/N249S/R318Y/ K63N/K82N/N84S/R150Y/ 5.3E+031.8E+03 34% 4,468 10 R338E/T343R/R403E/E410N R170E/T175R/R233E/E240NY155F/K228N/K247N/N249S/ Y[155]F/K63N/K82N/N84S/ 2.2E+03 1.4E+03 62%10,758 4 R318Y/R338E/T343R/R403E/ R150Y/R170E/T175R/R233E/ E410N E240NY155F/R338E/T343R/R403E Y[155]F/R170E/T175R/R233E 9.3E+04 1.2E+04 13%257 4 R338E/T343R/R403E R170E/T175R/R233E 1.9E+05 7.1E+02 0% 125 2Y155F/R338E/T343R/R403E/ Y[155]F/R170E/T175R/R233E/ 2.2E+05 2.6E+04 12%110 6 E410S E240S Y155F/N260S/R338E/T343R/ Y[155]F/N95S/R170E/T175R/4.0E+04 7.6E+03 19% 601 4 R403E R233E Y155F/I251S/R338E/T343R/Y[155]F/I86S/R170E/T175R/ 1.6E+05 1.5E+04 9% 146 2 R403E R233ER318Y/R338E/T343R/R403E/ R150Y/R170E/T175R/R233E/ 9.9E+03 2.9E+03 30%2,417 22 E410S E240S Y155F/K247N/N249S/T343R/ Y[155]F/K82N/N84S/T175R/1.4E+05 2.3E+04 16% 168 4 R403E R233E Y155F/K247N/N249S/R318Y/Y[155]F/K82N/N84S/R150Y/ 2.3E+03 1.7E+02 8% 10,415 2 R338E/T343R/R403ER170E/T175R/R233E K247N/N249S/R318Y/R338E/ K82N/N84S/R150Y/R170E/1.7E+03 2.0E+02 12% 14,156 4 T343R/R403E T175R/R233EY155F/K247N/N249S/R338E/ Y[155]F/K82N/N84S/R170E/ 8.9E+04 1.1E+04 13%268 4 T343R/R403E/E410N T175R/R233E/E240N K247N/N249S/R338E/T343R/K82N/N84S/R170E/T175R/ 8.6E+04 1.1E+04 13% 276 4 R403E/E410N R233E/E240NY155F/K247N/N249S/R318Y/ Y[155]F/K82N/N84S/R150Y/ 2.7E+04 1.4E+04 50%889 4 R338E R170E Y155F/K247N/N249S/R318Y/ Y[155]F/K82N/N84S/R150Y/4.0E+05 2.9E+05 72% 60 8 T343R T175R Y155F/K247N/N249S/R318Y/Y[155]F/K82N/N84S/R150Y/ 2.1E+03 5.3E+01 2% 11,125 2 R403E R233EY155F/K247N/N249S/R318Y/ Y[155]F/K82N/N84S/R150Y/ 1.3E+05 9.5E+04 75%188 6 E410N E240N Y155F/K247N/N249S/R338E/ Y[155]F/K82N/N84S/R170E/1.3E+04 1.0E+03 8% 1,819 2 R403E R233E Y155F/K247N/N249S/R338E/Y[155]F/K82N/N84S/R170E/ 1.2E+07 6.2E+06 51% 2 4 T343R T175RY155F/K247N/N249S/R318Y/ Y[155]F/K82N/N84S/R150Y/ 2.2E+05 1.0E+05 45%107 4 R338E/T343R/E410N R170E/T175R/E240N K247N/N249S/R318Y/R338E/K82N/N84S/R150Y/R170E/ 2.1E+05 8.2E+04 39% 114 4 T343R/E410N T175R/E240NY155F/K247N/N249S/R318Y/ Y[155]F/K82N/N84S/R150Y/ 2.8E+04 5.6E+03 20%842 4 T343R/R403E/E410N T175R/R233E/E240N K247N/N249S/R318Y/T343R/K82N/N84S/R150Y/T175R/ 2.5E+04 8.0E+03 32% 962 6 R403E/E410N R233E/E240NY155F/K247N/N249S/R338E/ Y[155]F/K82N/N84S/R170E/ 2.9E+06 2.2E+06 77% 86 E410N E240N Y155F/K247N/N249S/R318Y/ Y[155]F/K82N/N84S/R150Y/ 1.2E+041.0E+03 9% 2,011 4 T343R/R403E T175R/R233E K247N/N249S/R318Y/T343R/K82N/N84S/R150Y/T175R/ 9.8E+03 2.5E+03 26% 2,430 12 R403E R233EY155F/K247N/N249S/R318Y/ Y[155]F/K82N/N84S/R150Y/ 3.6E+05 1.2E+05 32% 664 T343R/E410N T175R/E240N K247N/N249S/R318Y/T343R/K82N/N84S/R150Y/T175R/ 4.9E+04 6.5E+03 13% 487 4 E410N E240NY155F/K247N/N249S/R338E/ Y[155]F/K82N/N84S/R170E/ 4.4E+04 1.1E+04 26%549 4 T343R/R403E T175R/R233E K247N/N249S/R338E/T343R/K82N/N84S/R170E/T175R/ 5.0E+04 1.7E+04 35% 482 4 R403E R233EK247N/N249S/R338E/T343R/ K82N/N84S/R170E/T175R/ 1.4E+07 7.2E+06 53% 2 5E410N E240N Y155F/K247N/N249S/T343R/ Y[155]F/K82N/N84S/T175R/ 6.2E+055.6E+04 9% 39 4 R403E/E410N R233E/E240N K247N/N249S/T343R/R403E/K82N/N84S/T175R/R233E/ 4.2E+05 8.1E+04 19% 58 4 E410N E240NY155F/R318Y/R338E/T343R Y[155]F/R150Y/R170E/T175R 4.4E+05 1.9E+05 43% 556 R318Y/R338E/T343R R150Y/R170E/T175R 1.8E+06 8.6E+05 48% 13 4Y155F/R318Y/T343R/R403E Y[155]F/R150Y/T175R/R233E 1.1E+04 9.1E+02 8%2,114 2 Y155F/T343R/R403E/E410N Y[155]F/T175R/R233E/E240N 8.8E+053.3E+03 0% 27 2 Y155F/K247N/N249S/R318Y/ Y[155]F/K82N/N84S/R150Y/3.7E+05 1.1E+05 28% 64 6 R338E/T343R R170E/T175RK247N/N249S/R318Y/R338E/ K82N/N84S/R150Y/R170E/ 3.2E+05 1.4E+05 44% 74 6T343R T175R Y155F/K247N/N249S/T343R/ Y[155]F/K82N/N84S/T175R/ 3.5E+064.8E+05 14% 7 2 E410N E240N Y155F/K247N/N249S/R403E/Y[155]F/K82N/N84S/R233E/ 1.3E+05 3.3E+04 26% 191 14 E410N E240NY155F/R338E/T343R/E410N Y[155]F/R170E/T175R/E240N 1.3E+07 1.0E+07 78% 26 R338E/T343R/E410N R170E/T175R/E240N 2.0E+07 6.3E+06 31% 1 4Y155F/R318Y/T343R/E410N Y[155]F/R150Y/T175R/E240N 2.0E+05 5.9E+04 29%118 4 R318Y/T343R/E410N R150Y/T175R/E240N 1.2E+06 1.1E+05 9% 20 2K228N/R150Y/R338E/T343R/ K63N/R150Y/R170E/T175R/ 7.1E+03 3.3E+02 5%3,343 2 R403E/E410N R233E/E240N K228N/K247N/N249S/R318Y/K63N/K82N/N84S/R150Y/ 1.0E+03 2.3E+02 22% 23,389 2 R338E/T343R/R403ER170E/T175R/R233E K228N/247N/N249S/R318Y/ K63N/K82N/N84S/R150Y/ 6.3E+051.0E+05 17% 38 2 R338E/T343R/E410N R170E/T175R/E240NK228N/K247N/N249S/R318Y/ K63N/K82N/N84S/R150Y/ 1.7E+04 2.4E+03 14% 1,4222 T343R/R403E/E410N T175R/R233E/E240N

Example 6 Pharmacokinetic and Pharmacodynamic Analysis of FIXaPolypeptides

The pharmacokinetic (PK) and pharmacodynamic (PD) properties of the FIXavariant polypeptides were assessed by measuring the amount of variantFIX in mouse plasma at various timepoints following intravenousadministration. Two assays were used to quantify FIXa in plasma. AnELISA was used to quantify total FIX protein in mouse plasma to assessthe pharmacokinetic properties, and a FIX-dependant clotting assay(activated partial thromboplastin time (aPTT) assay using FIX-depletedplasma) was used to quantify the coagulant activity of the FIXpolypeptides in plasma, thus assessing the pharmacodynamic properties.

Animals.

Male CD-1 mice (30-40 gm), supplied by Charles River Laboratories(Hollister, Calif.) were quarantined for at least 3 days beforetreatment. For serial PK studies, male CD-1 mice (30-37 gm) were fittedwith an indwelling jugular vein cannula. Filtered tap water and food wasavailable ad libitum prior to use in PD or PK experiments.

A. Dosing and Blood Collection

Mice (N=3 per time point) were administered the FIX polypeptidesintravenously (˜1.4 mg/kg for PK studies and ˜400 IU/kg for PD studies,dose volume 2 ml/kg) via the tail vein. At the appropriate time afterdosing, animals were anesthetized and blood was drawn (0.5-1 mL) usingterminal cardiac puncture into syringes containing citrate. In someexperiments where insufficient amount of protein was available, a totalof only 4-6 animals were used for serial bleeding at staggered timepoints; two mice were used for each full time course in order to collectall time points without removing excess blood volume. Blood was sampledin restrained conscious animals by first removing a small amount ofblood into a 0.1 mL syringe containing 0.9% saline. A syringe containing4.5 μl of 0.1M sodium citrate was then attached and 0.05 mL blood waswithdrawn into the syringe and the blood was transferred to a 1.5 mLtube. The initial syringe was reattached and 0.07 mL of saline pushedback through the cannula. The cannula was capped until the next timepoint, when the process was repeated. For all studies, blood sampleswere centrifuged within 15 minutes of collection (9000 rpm, 8 minutes,4° C.) and the plasma removed and immediately flash frozen in liquidnitrogen and then stored frozen (−70° C.) pending analysis.

A. PK Assessment.

Citrated blood samples were collected at various times up to 1440 minpost dose (i.e., Predose, 2, 4, 10, 30, 60, 120, 240, 360, 480, 960 and1440 min) by cardiac puncture for terminal experiments or indwellingcatheter for serial experiments. Plasma concentrations of rFIX weredetermined using a factor IX specific ELISA utilizing a matched pair ofdetection and capture antibodies (#FIX-EIA, Affinity Biologicals,Ancaster, ON). Briefly, an affinity purified polyclonal antibody to FIXis coated onto the wells of a plate. The plates are washed and plasmasamples containing FIX are applied. Plasma samples are diluted 1:750 and1:1500 on the plate. After washing the plate to remove unbound material,a peroxidase conjugated detection antibody to FIX is added to the plateto bind to the captured FIX. After washing the plate to remove unboundconjugated antibody, the peroxidase activity is expressed by incubationwith chemiluminescent substrate and read at 425 nM on an EnVision platereader. The standard curve is linear over the entire concentration rangeand spans the concentrations of 0.82 pg/ml to 30 ng/ml. The FIX variantitself is used for the standard curve to eliminate differences in theantibody affinity. Each sample is measured on two separate assay platesand those measurements within the range of the standard curve are usedto calculate the concentration of FIX variants in the plasma sample.

PD Assessment.

The plasma pharmacodynamic activity of rFIX was quantified using anactivated partial thromboplastin time (aPTT) assay and FIX deficienthuman plasma (STACLOT C.K. PREST kit, Diagnostica Stago, Asnieres,France) per the manufacturer's instructions. Briefly, the aPTT assayinvolves the recalcification of plasma in the presence of cephalin(platelet substitute) and activator (koalin). Using FIX deficient humanplasma, the aPTT assay is specific for FIX. The aPTT assay was performedas described in the manufacturers' product insert. Briefly, citratedblood samples were collected at the same time points described for PKassessment. Plasma samples were diluted 1:100 in Tris buffered salinecontaining 0.1% bovine serum albumin (Probumin, Millipore, Billerica,Mass.). Diluted plasma or standard was combined with FIX deficient humanplasma and cephalin/kaolin reagent and incubated for 180 seconds.Coagulation was initiated by the addition of calcium (CaCl₂).Coagulation time in seconds was measured using an STArt4 instrument(Diagnostica Stago, Asnieres, France). Using a standard curve made fromknown concentrations of rFIX, plasma FIX concentrations wereinterpolated from the log concentration VS. log time standard curve plotand then background FIX activity (from pre dose animals) was subtracted.The lower limit of quantification for factor IX activity was ˜10 ng/mL.

PD and PK Data Analysis.

PD (aPTT) and PK (ELISA) parameters from mouse studies with rFIXvariants were calculated using non compartmental analysis in WinNonLin(v5.1, Pharsight Corp., Mountain View, Calif.). Both the PD and PK ofrFIX variants followed apparent biexponential plasma decay. Selectparameters for each variant tested are provided in Table 25 for PD(using the aPTT assay) and Tables 26-27 for PK (using the ELISA assay).Table 26 reflects data for additional FIXa variants and provide newoverall averages calculated to include additional experimentalreplicates (n) for FIXa variants in Table 26. The PD parameters includedhalf-life (terminal, min), MRT (MRT_(0-inf), min), Area under the curve(AUC) 0-last (min·μg/mL)/Dose (mg/kg); Maximal concentration (C_(max);(μg/mL)/Dose (μg/kg), Vd (mL/kg) and Clearance (Cl, mL/min/kg).

Definitions and Formulae Used to Calculate Pharmacokinetic Parameters.

Plasma half-life (the half life of the FIX polypeptide during theterminal phase of plasma FIX concentration-versus-time profile; T_(1/2β)(calculated as −ln 2 divided by the negative slope during the terminalphase of the log-linear plot of the plasma FIX concentration-versus-timecurve); MRT_(0-last) is the mean time the FIX polypeptide resides inbody; calculated as AUMC_(0-last)/AUC_(0-last), where AUMC_(0-last) isthe total area under the first moment-versus-time curve and AUC asdescribed subsequently); AUC_(0-last)/Dose is calculated as[AUC_((0-t))], where t is the last time point with measurable plasmaconcentration of the FIX polypeptide divided by the IV dose (mg/kg);AUC_(0-inf)/Dose is calculated as [AUC_((0-t))+Ct/(ln 2/T_(1/2β))] wheret is the last time point with measurable plasma concentration of the FIXpolypeptide divided by the IV dose (mg/kg); C_(max)/Dose (ug/mL permg/kg), where C_(max) is the time post dose corresponding to the maximalmeasured plasma FIX concentration; Cl is systemic clearance calculatedas (Dose/AUC_(0-inf)); V_(ss) is the steady state volume ofdistribution; calculated as MRT*Cl; and V_(z) is the volume ofdistribution based on the terminal elimination constant (β); calculatedas Cl/(ln 2/T_(1/2β)).

TABLE 25 PD properties of FIX variants assessed by aPTT assay Mutation(Mature FIX C_(max)/ numbering) N T_(1/2β) MRT_(0-inf) dose AUC_(0-inf)Cl Vz Vss BeneFIX ® Coagulation FIX 2 296 354 19.3 2641 0.41 169 142(T148A)

TABLE 26 PK properties of FIX variants assessed by ELISA AUC/ AUC/ MRTCmax/ Dose Dose Mutation N T½_(β) 0-inf Dose 0-last 0-inf Vz ClBeneFIX ® Coagulation FIX 3 314 ± 128 366 ± 105  9.1 ± 1.5 1298 ± 2981522 ± 158  308 ± 160 0.74 ± 0.06 (T148A) T148A 8 383 ± 109 435 ± 12810.2 ± 2.1 1620 ± 195 1747 ± 234 317 ± 82 0.58 ± 0.08 CatalystBiosciences WT 2 329 360 11.9 2036 2121 229 0.48 A103N/N105S 2 375 48112.5 2841 3068 177 0.33 D104N/K106S 2 428 558 13.9 3379 3786 164 0.26K106N/V108S 2 510 629 12.8 2748 3202 234 0.32 D85N 2 528 607 9.5 17982046 372 0.49 D64N 2 447 519 11.8 1933 2152 304 0.47 D64A 2 364 372 11.51351 1466 359 0.68 N167D 2 334 318 8.9 1129 1176 410 0.85 N167Q 3 337 ±8.8  323 ± 4.2   8.2 ± 1.2 1495 ± 258 1554 ± 268 318 ± 42 0.66 ± 0.10S61A 2 397 412 10.0 1685 1800 325 0.57 S53A 2 382 462 11.2 2146 2321 2380.43 T159A 2 232 227 10.5 1036 1048 315 0.97 T169A 2 348 319 8.3 836 889567 1.15 T172A 3 494 ± 187 571 ± 214 11.2 ± 2.9 2055 ± 408 2366 ± 676295 ± 31 0.45 ± 0.13 T179A 2 377 431 12.5 2291 2458 223 0.42 Y155H 2 465552 11.6 2365 2638 253 0.38 Y155Q 1 552 645 13.6 2583 3045 262 0.33S158E 2 433 471 14.5 2029 2222 291 0.46 N157Q 2 335 352 11.3 1185 1238395 0.83 N157D 2 290 265 9.9 1166 1211 393 0.93 Y155F 2 443 567 18.13941 4375 149 0.23 A103N/N105S/Y155F 2 562 619 13.1 2427 2496 325 0.40D104N/K106S/Y155F 3 514 ± 80  581 ± 81  13.8 ± 1.0  3057 ± 1032 3181 ±989 243 ± 47 0.34 ± 0.13 D203N/F205T 3 481 ± 69  566 ± 29   9.4 ± 1.92028 ± 448 2289 ± 489 314 ± 91 0.45 ± 0.09 D203N/F205T/D85N 1 291 40612.4 1538 2044 205 0.49 K228N/D85N 2 459 565 11.3 2616 2926 227 0.35K228N/A103N/N105S 2 583 701 14.4 3032 3301 255 0.30 K228N/D104N/K106S 2801 913 13.6 2050 2238 513 0.45 K228N/Y155F 2 626 679 8.6 2073 2149 4310.47 K228N/D104N/K106S/Y155F 2 551 614 14.0 3730 3822 211 0.27 I251S 2565 718 10.1 2646 3137 260 0.32 I251S/A103N/N105S 2 444 542 14.3 24452719 241 0.38 I251S/D104N/K106S 2 692 802 13.9 2533 2664 375 0.38I251S/Y155F 2 572 660 12.2 2591 2790 291 0.37 A262S 3 373 ± 87  453 ±91  14.4 ± 3.8 2716 ± 732 2926 ± 908 188 ± 29 0.36 ± 0.10 E410N* 2 439551 7.4 893 1365 469 0.75 E239N 2 338 416 10.7 1657 1908 257 0.54K247N/N249S 6 627 ± 174 734 ± 244 10.8 ± 3.4 2196 ± 737 2545 ± 795  387± 154 0.42 ± 0.11 Y155F/K247N/N249S 2 538 608 10.6 1752 1880 420 0.53K247N/N249S/A103N/N105S 2 736 852 21.5 4369 4699 226 0.21K247N/N249S/D104N/K106S/ 2 603 714 16.8 3744 3889 233 0.27 Y155FS319N/L321S 2 351 427 11.4 2270 2409 210 0.42 N260S 3 496 ± 157 619 ±170 11.5 ± 3.8  3364 ± 1300  3687 ± 1457  231 ± 156 0.30 ± 0.11D104N/K106S/N260S 2 805 1001 16.1 4736 5248 220 0.20 Y155F/N260S 2 607682 18.4 3408 3530 257 0.27 Y284N 2 400 478 9.0 2052 2210 270 0.46R318Y/E410N 1 428 474 6.1 575 686 900 1.46 R338E/E410N 2 334 376 6.2 718844 570 1.18 R338E/R403E/E4100N 5 436 ± 24  507 ± 29  13.4 ± 2.0 3052 ±522 3302 ± 656 196 ± 49 0.31 ± 0.06 D203N/F205T/E240N 2 600 679 6.8 671799 1080 1.25 D203N/F205T/R338E 2 307 419 9.3 1186 1586 281 0.63D203N/F205T/R338A 2 317 403 9.0 1063 1397 327 0.72 D203N/F205T/R318Y 2258 286 8.7 508 601 732 1.91 D203N/F205T/R338E/R403E 2 303 419 11.3 21052804 156 0.36 K228N/E410N 2 373 479 6.0 721 1025 522 0.98 K228N/R338E 2248 340 10.4 1403 1736 207 0.58 R318Y/R338E/E410N 5 424 ± 306 515 ± 378 5.8 ± 1.6  645 ± 310  774 ± 454  778 ± 272  1.6 ± 0.73R318Y/R338E/E410N/D104N/ 2 502 531 8.9 2008 2041 355 0.49 K106SR318Y/R338E/E410N/Y155F 2 555 584 6.5 678 721 1136 1.53K228N/R318Y/E410N 1 304 408 6.0 686 906 485 1.10 R318Y/R338E/R403E/E410N5 442 ± 22  534 ± 28  16.4 ± 3.7 3902 ± 867 4232 ± 996 157 ± 38 0.25 ±0.05 A103N/N105S/R318Y/R338E/ 2 421 527 16.2 3605 3935 157 0.26R403E/E410N D104N/K106S/R318Y/R338E/ 2 417 517 15.1 3114 3392 183 0.30R403E/E410N Y155F/R318Y/R338E/R403E/ 2 565 649 12.4 3687 3772 226 0.27E410N R318Y/R338E/R403E/E410N/ 3 669 ± 145 819 ± 223 17.2 ± 2.0  5844 ±1064  6204 ± 1393  156 ± 8.7 0.17 ± 0.04 A103N/N105S/Y155FR318Y/R338E/R403E/E410N/ 2 472 575 14.4 5885 5967 114 0.17D104N/K106S/Y155F D203N/F205T/R318Y/E410N 1 431 475 8.0 637 761 816 1.31R338L 2 368 377 11.2 1761 1861 285 0.54 K316M 2 527 665 7.9 1846 2142356 0.47 E239S 2 462 542 11.3 2184 2416 278 0.41 E239A 2 538 544 13.11973 2209 353 0.45 E239R 2 431 709 8.9 1668 2020 307 0.50 E239K 2 400370 14.4 2107 2222 278 0.48 H257F 2 328 357 10.3 1689 1820 273 0.70H257Y 2 352 353 13.6 1971 2063 245 0.49 H257E 2 491 520 10.9 2185 2411294 0.42 H257S 2 435 511 8.2 1630 1769 358 0.57 T412A 2 473 539 7.1 15611756 379 0.58 T412V 2 579 665 8.3 1258 1454 565 0.69 E410N/T412A 2 461514 2.8 364 398 1679 2.51 E410N/T412V 2 340 390 3.7 431 487 906 2.27E410Q 2 276 283 7.2 445 484 836 2.19 E410S 2 310 286 7.2 753 775 5871.32 E410A 2 363 328 8.6 528 554 946 1.81 E410D 2 348 377 9.2 1473 1596320 0.63 N346D 2 349 395 13.3 2817 2956 170 0.34 Y155F/N346D 2 472 47817.0 3934 3986 176 0.26 N346Y 2 329 325 11.7 1246 1297 365 0.77 Y345T 2359 453 6.1 1124 1200 438 0.85 T343R 2 402 504 6.5 1143 1234 487 0.85T343E 2 414 461 12.6 1740 1877 318 0.53 T343Q 2 434 442 9.0 1626 1737408 0.63 F342I 2 400 476 8.3 1133 1224 491 0.88 T343R/Y345T 2 325 3249.1 1094 1130 422 0.90 R318Y/R338E 2 340 313 11.2 1402 1452 336 0.69K228N/I251S 2 586 657 11.3 1473 1588 551 0.65 K228N/R318Y/R338E/R403E/ 2476 647 9.1 2400 2726 261 0.37 E410N K228N/R318Y/R338E/R403E/ 3 615 ±135 750 ± 191 18.6 ± 2.1  5496 ± 2044  5970 ± 2260 158 ± 50 0.18 ± 0.06E410N/Y155F K228N/R318Y/R338E/R403E/ 2 587 713 24.8 6153 6725 125 0.15E410N/D85N I251S/R318Y/R338E/R403E/ 3 412 ± 140 542 ± 181 15.7 ± 4.92306 ± 884  2636 ± 1261 242 ± 89 0.44 ± 0.17 E410ND104N/K106S/I251S/R318Y/ 4 687 ± 60  874 ± 82  17.2 ± 2.2 7653 ± 4568127 ± 520 122 ± 10 0.12 ± 0.01 R338E/R403E/E410NI251S/R318Y/R338E/R403E/ 2 492 620 19.9 5704 6510 110 0.15 E410N/Y155FI251S/R318Y/R338E/E410N 2 591 630 7.5 1245 1292 664 0.78D104N/K106S/D104N/K106S/ 2 726 819 16.4 1512 1612 650 0.62I251S/R318Y/R338E/E410N/ K247N/N249S/R318Y/R338E/ 2 637 807 15.4 52835541 170 0.18 R403E/E410N Y155F/K247N/N249S/R318Y/ 2 613 758 13.8 53355549 160 0.18 R338E/R403E/E410N A103N/N105S/K247N/N249S/ 2 615 783 18.67319 7612 117 0.13 R318Y/R338E/R403E/E410N D104N/K106S/K247N/N249S/ 2626 754 19.4 6332 6580 140 0.15 R318Y/R338E/R403E/E410NK228N/N84S/R318Y/R338E/ 2 512 539 18.1 1925 1967 396 0.54 E410NY155F/K228N/N84S/R318Y/ 2 617 685 8.1 1170 1221 745 0.83 R338E/E410NR318Y/R338E/R403E/E410S 2 382 395 14.7 2897 2971 184 0.34R318Y/R338E/E410S 2 356 326 7.7 488 511 1066 2.08 K228N/K247N/N249S 2662 753 19.6 3390 3578 268 0.28 K228N/K247N/N249S/D104N/ 3 781 ± 55  939± 48  18.5 ± 3.8  6111 ± 1900  6606 ± 1949 183 ± 63 0.16 ± 0.04K106S/Y155F K228N/K247N/N249S/D104N/ 2 758 838 17.9 3792 4035 271 0.25K106S K228N/K247N/N249S/Y155F 2 549 643 17.2 3002 3269 246 0.31I251S/R318Y/R338E/R403E/ 3 599 ± 89  753 ± 121 21.7 ± 3.2  8567 ± 2834 9233 ± 2860  96.6 ± 15.4 0.11 ± 0.03 E410N/Y155FR318Y/R338E/R403E/E410N/ 2 424 456 20.0 4730 4892 124 0.20 T412VR318Y/R338E/R403E/E410N/ 2 380 439 17.5 4994 5115 107 0.20 T412AR318Y/R338E/R403E/T412A 3 399 ± 88  477 ± 108 19.7 ± 0.7  4320 ± 2385 4505 ± 2357 144 ± 48 0.27 ± 0.15 R318Y/R3380E/T412A 2 462 401 13.6 16741691 398 0.60 N260S/R318Y/R338E/R403E/ 2 583 743 23.9 6821 7488 111 0.13E410N D104N/K106S/N260S/R318Y/ 2 779 999 17.2 7100 7728 145 0.12R338E/R403E/E410N Y155F/N260S/R318Y/R338E/ 2 628 758 21.4 5214 5465 1670.21 R403E/E410N R318Y/R338E/N346D/R403E/ 2 474 575 25.2 7623 8140 860.12 E410N Y155F/R318Y/R338E/N346D/ 2 540 641 18.2 5039 5172 154 0.20R403E/E410N K247N/N249S/N260S 2 549 632 17.4 4156 4262 186 0.23Y155F/K247N/N249S/N260S 2 691 814 24.0 3857 4085 244 0.22D104N/K106S/K247N/N249S/ 2 712 859 16.5 4187 4458 235 0.23 N260SD104N/K106S/Y155F/K247N/ 2 680 856 23.3 7026 7423 134 0.14 N249S/N260SK247N/N249S/N260S/R318Y/ 2 691 875 18.9 6353 6737 149 0.13R338E/R403E/E410N R318Y/R338E/T343R/R403E/ 2 531 560 20.5 3766 3862 2000.27 E410N R338E/T343R 2 534 453 12.8 798 813 949 1.23 *80% glycosylatedform of E410N

TABLE 27 PK properties of FIX variants assessed by ELISA MutationMutation AUC/ AUC/ (Mature FIX (Chymotrypsin Terminal Dose Dose MRTNumbering) Numbering) N T½ (0-last) (0-inf) (0-inf) N157D N[157]D 2 2901166 1211 265 Y155F Y[155]F 2 443 3941 4375 567 A103N/N105S/A[103]N/N[105]S/ 2 562 2427 2496 619 Y155F Y[155]F D104N/K106S/D[104]N/K[106]S/ 3 514 ± 79.8  3060 ± 1030 3180 ± 989 581 ± 81.0 Y155FY[155]F WT Catalyst 2 329 2036 2121 360 Biosciences WT A103N/N105SA[103]N/N[105]S 2 375 2841 3068 481 D104N/K106S D[104]N/K[106]S 2 4283379 3786 558 K106N/V108S K[106]N/V[108]S 2 510 2748 3202 629 575 ± 89.31530 ± 321  1680 ± 83.3 623 ± 83.3 T148A BeneFIX, 3 314 ± 128 1300 ± 2981520 ± 158 366 ± 105  T[148]A T148A T[148]A 8 383 ± 109  1620 ± 195 1750± 234 435 ± 128  271 1548 1583 311 D64N D[64]N 2 447 1933 2152 519 D64AD[64]A 2 364 1351 1466 372 N167D N[167]D 2 334 1129 1176 318 N167QN[167]Q 3 337 ± 8.75 1500 ± 258 1550 ± 268 323 ± 4.25 S61A S[61]A 2 3971685 1800 412 S53A S[53]A 2 382 2146 2321 462 T159A T[159]A 2 232 10361048 227 T169A T[169]A 2 348 836 889 319 494 ± 187  2050 ± 408  237 ±676 571 ± 214  377 2291 2458 431 Y155H Y[155]H 2 465 2365 2638 552 Y155QY[155]Q 1 552 2583 3045 645 S158E S[158]E 2 433 2029 2222 471 N157QN[157]Q 2 335 1185 1238 352 481 ± 69.0 2030 ± 448 2290 ± 489 566 ± 28.6291 1538 2044 406 F205T D39N/F41T 490 ± 57.8 2340 ± 519 2570 ± 682 570 ±27.9 583 3032 3301 701 K228N N[105]S/K63N D104N/K106S/ D[104]N/ 2 8012050 2238 913 K228N K[106]S/K63N Y155F/K228N Y[155]F/K63N 2 626 20732149 679 D104N/K106S/ D[104]N/K[106]S/ 2 551 3730 3822 614 Y155F/K228NY[155]F/K63N I251S I86S 2 565 2646 3137 718 A103N/N105S/ A[103]N/ 2 4442445 2719 542 I251S N[105]S/I86S D104N/K106S/ D[104]N/ 2 692 2533 2664802 I251S K[106]S/I86S Y155F/I251S Y[155]F/I86S 2 572 2591 2790 660A262S A95bS 2 373 2716 2926 453 E410N E240N 2 439 893 1365 551 E239NE74N 2 338 1657 1908 416 627 ± 174  2200 ± 737 2540 ± 795 734 ± 244  5381752 1880 608 N249S A103N/N105S/ A[103]N/N[105]S/ 2 736 4369 4699 852K247N/N249S K82N/N84S D104N/K106S/ D[104]N/K[106]S/ 2 571 2052 2109 632K247N/N249S K82N/N84S D104N/K106S/ D[104]N/K[106]S/ 2 603 3744 3889 714Y155F/K247N/ Y[155]F/ N249S K82N/N84S S319N/L321S S151N/L153S 2 351 22702409 427 496 ± 157  3360 ± 1300  3690 ± 1460 619 ± 170  805 4736 52481001 N260S K[106]S/N95S Y155F/N260S Y[155]F/N95S 2 607 3408 3530 682Y284N Y117N 2 400 2052 2210 478 R318Y/E410N R150Y/E240N 1 428 575 686474 R338E/E410N R170E/E240N 2 334 718 844 376 436 ± 24.4 3050 ± 522 3300± 656 507 ± 28.9 E410N E240N 600 671 799 679 E410N E240N D203N/F205T/D39N/F41T/ 2 307 1186 1586 419 R338E R170E D203N/F205T/ D39N/F41T/ 2 3171063 1397 403 R338A R170A D203N/F205T/ D39N/F41T/ 2 258 508 601 286R318Y R150Y D203N/F205T/ D39N/F41T/ 2 303 2105 2804 419 R338E/R403ER170E/R233E K228N/E410N K63N/E240N 2 373 721 1025 479 K228N/R338EK63N/R170E 2 248 1403 1736 340 R318Y/R338E/ R150Y/E240N/ 5 424 ± 306  645 ± 310  774 ± 454 515 ± 378  E410N R170E 502 2008 2041 531R318Y/R338E/ R150Y/R170E/ E410N E240N/ Y155F/R318Y/ Y[155]F/R150Y/ 2 555678 721 584 R338E/E410N R170E/E240N K228N/R318Y/ K63N/R150Y/ 1 304 686906 408 E410N E240N 442 ± 22.1 3900 ± 867 4230 ± 996 534 ± 28.0R403E/E410N R233E/E240N 421 3605 3935 527 R318Y/R338E/ R150Y/R170E/R403E/E410N R233E/E240N D104N/K106S/ D[104]N/K[106]S/ 2 417 3114 3392517 R318Y/R338E/ R150Y/R170E/ R403E/E410N R233E/E240N Y155F/R318Y/Y[155]F/R150Y/ 2 565 3687 3772 649 R338E/R403E/ R170E/R233E/ E410N E240N669 ± 145   5840 ± 1060  6200 ± 1390 819 ± 223  Y155F/R318Y/N[105]S/Y[155]F/ R338E/R403E/ R150Y/R170E/ E410N R233E/E240N 472 58855967 575 Y155F/R318Y/ K[106]S/Y[155]F/ R338E/R403E/ R150Y/R170E/ E410NR233E/E240N D203N/F205T/ D39N/F41T/ 1 431 637 761 475 R318Y/E410NR150Y/E240N R338L R170L 2 368 1761 1861 377 K316M K148M 2 527 1846 2142665 E239S E74S 2 462 2184 2416 542 E239A E74A 2 538 1973 2209 544 E239RE74R 2 431 1668 2020 709 E239K E74K 2 400 2107 2222 370 H257F H92F 2 3281689 1820 357 H257Y H92Y 2 352 1971 2063 353 H257E H92E 2 491 2185 2411520 H257S H92S 2 435 1630 1769 511 T412A T242A 2 473 1561 1756 539 T412VT242V 2 579 1258 1454 665 E410N/T412A E240N/T242A 2 461 364 398 514E410N/T412V E240N/T242V 2 340 431 487 390 E410Q E240Q 2 276 445 484 283E410S E240S 2 310 753 775 286 E410A E240A 2 363 528 554 328 E410D E240D2 348 1473 1596 377 N346D N178D 2 349 2817 2956 395 Y155F/N346D178D/Y[155]F 2 472 3934 3986 478 N346Y N178Y 2 329 1246 1297 325 Y345TY177T 2 359 1124 1200 453 T343R Y175R 2 402 1143 1234 504 T343E T175E 2414 1740 1877 461 T343Q Y175Q 2 434 1626 1737 442 F342I F174I 2 400 11331224 476 T343R/Y345T T175R/Y177T 2 325 1094 1130 324 R318Y/R338ER150Y/R170E 2 340 1402 1452 313 K228N/I251S K63N/I86S 2 586 1473 1588657 K228N/R318Y/ K63N/R150Y/ 2 476 2400 2726 647 R338E/R403E/R170E/R233E/ E410N E240N Y155F/K228N/ Y[155]F/K63N/ 3 615 ± 135   5500 ±2040  5970 ± 2260 750 ± 191  R318Y/R338E/ R150Y/R170E/ R403E/E410NR233E/E240N D85N/K228N/ D[85]N/K63N/ 2 587 6153 6725 713 R318Y/R338E/R150Y/R170E/ R403E/E410N R233E/E240N I251S/R318Y/ I86S/R150Y/ 3 412 ±140  2310 ± 884  2640 ± 1260 542 ± 181  R338E/R403E/ R170E/R233E/ E410NE240N D104N/K106S/ D[104]N/K[106]S/ 4 687 ± 60.2 7650 ± 456 8130 ± 520874 ± 81.7 I251S/R318Y/ I86S/R150Y/ R338E/R403E/ R170E/R233E/ E410NE240N 492 5704 6510 620 R318Y/R338E/ R150Y/R170E/ R403E/E410NR233E/E240N I251S/R318Y/ I86S/R150Y/ 2 591 1245 1292 630 R338E/E410NR170E/E240N D104N/K106S/ D[104]N/K[106]S 2 726 1512 1612 819I251S/R318Y/ I86S/R150Y/ R338E/E410N R170E/E240N K247N/N249S/ K82N/N84S/2 637 5283 5541 807 R318Y/R338E/ R150Y/R170E/ R403E/E410N R233E/E240NY155F/K247N/ Y[155]F/ 2 613 5335 5549 758 N249S/R318Y/ K82N/N84S/R338E/R403E/ R150Y/R170E/ E410N R233E/E240N A103N/N105S/ A[103]N/N[105]S2 615 7319 7612 783 K247N/N249S/ K82N/N84S/ R318Y/R338E/ R150Y/R170E/R403E/E410N R233E/E240N/ D104N/K106S/ D[104]N/K[106]S 2 626 6332 6580754 K247N/N249S/ K82N/N84S/ R318Y/R338E/ R150Y/R170E/ R403E/E410NR233E/E240N/ D104N/K106S/ D[104]N/K[106]S/ 2 846 8069 8807 1020Y155F/K247N/ Y[155]F/K82N/ N249S/R318Y/ N84S/R150Y/ R338E/R403E/R170E/R233E/ E410N E240N K247N/N249S/ K82N/N84S/ 2 512 1925 1967 539R318Y/R338E/ R150Y/R170E/ E410N E240N Y155F/K247N/ Y[155]F/ 2 617 11701221 685 N249S/R318Y/ K82N/N84S/ R338E/E410N R150Y/R170E/ E240N/R318Y/R338E/ R150Y/R170E/ 2 382 2897 2971 395 R403E/E410S R233E/E240SR318Y/R338E/ R150Y/R170E/ 2 356 488 511 326 E410S E240S K228N/K247N/K63N/K82N/ 2 662 3390 3578 753 N249S N84S 781 ± 55.2  6110 ± 1900  6610± 1950 939 ± 48.2 Y155F/K228N/ Y[155]F/K63N/ K247N/N249S K82N/N84S 7583792 4035 838 K228N/K247N/ K63N/K82N/ N249S N84S Y155F/K228N/Y[155]F/K63N/ 2 549 3002 3269 643 K247N/N249S K82N/N84S 599 ± 88.6  8570± 2830  9230 ± 2860 753 ± 120  N249S/R318Y/ R150Y/R170E/ R338E/R403E/R233E/E240N/ E410N D104N/K106S/ D[104]N/K[106]S/ 3 806 ± 88.6  9330 ±2830  9990 ± 2860 912 ± 120  K228N/K247N/ K63N/K82N/ N249S/R318Y/N84S/R150Y/ R338E/R403E/ R170E/R233E/ E410N E240N 559 10704 11042 710K247N/N249S/ K82N/N84S/ R318Y/R338E/ R150Y/R170E/ R403E/E410NR233E/E240N R318Y/R338E/ R150Y/R170E/ 2 424 4730 4892 456 R403E/E410N/R233E/E240N/ T412V T242V R318Y/R338E/ R150Y/R170E/ 2 380 4994 5115 439R403E/E410N/ R233E/E240N/ T412A T242A R318Y/R338E/ R150Y/R170E/ 3 399 ±88.1  4320 ± 2380  4500 ± 2360 477 ± 108  R403E/T412A R233E/T242AR318Y/R338E/ R150Y/R170E/ 2 462 1674 1691 401 T412A T242A R318Y/R338E/150Y/R170E/ 2 251 524 555 226 E410N/T412V E240N/T242V N260S/R318Y/N95S/R150Y/ 2 583 6821 7488 743 R338E/R403E/ R170E/R233E/ E410N E240ND104N/K106S/ D[104]N/K[106]S/ 2 779 7100 7728 999 N260S/R318Y/N95S/R150Y/ R338E/R403E/ R170E/R233E/ E410N E240N Y155F/N260S/Y[155]F/N95S/ 2 628 5214 5465 758 R318Y/R338E/ R150Y/R170E/ R403E/E410NR233E/E240N R318Y/R338E/ R150Y/R170E/ 2 474 7623 8140 575 N346D/R403E/N178D/R233E/ E410N E240N Y155F/R318Y/ Y[155]F/R150Y/ 2 540 5039 5172 641R338E/N346D/ R170E/N178D/ R403E/E410N R233E/E240N K247N/N249S/K82N/N84S/ 2 549 4156 4262 632 N260S N95S Y155F/K247N/ Y[155]F/K82N/ 2691 3857 4085 814 N249S/N260S N84S/N95S D104N/K106S/ D[104]N/K[106]S/ 2712 4187 4458 859 K247N/N249S/ K82N/N84S/ N260S N95S D104N/K106S/D[104]N/K[106]S/ 2 680 7026 7423 856 Y155F/K247N/ Y[155]F/K82NN249S/N260S N84S/N95S/ K247N/N249S/ K82N/N84S/ 2 691 6353 6737 875N260S/R318Y/ N95S/R150Y/ R338E/R403E/ R170E/R233E/ E410N E240NY155F/K247N/ Y[155]F/K82N/ 1 1038 8401 9376 1068 N249S/N260S/ N84S/N95S/R318Y/R338E/ R150Y/R170E/ R403E/E410N R233E/E240N R318Y/R338E/T175R/R233E/ 2 531 3766 3862 560 T343R/R403E/ E240N/R150Y/ E410N R170EY155F/R318Y/ Y[155]F/R150Y/ 1 182 3223 4335 259 R338E/T343R/R170E/T175R/ R403E/E410N R233E/E240N D104N/K106S/ D[104]N/K[106]S/ 3 666± 89.9 7270 ± 729 7550 ± 708 699 ± 88.0 R318Y/R338E/ R150Y/R170E/T343R/R403E/ T175R/R233E/ E410N E240N R338E/T343R R170E/T175R 2 534 798813 453 T343R/N346Y T175R/N178Y 3 276 ± 19.9 1080 ± 331 1100 ± 333 228 ±7.76 R318Y/R338E/ R150Y/R170E/ 2 324 2394 2487 335 N346Y/R403E/N178Y/R233E/ E410N E240N 303 3569 3691 329 T343R/N346Y/ T175R/N178Y/R403E/E410N R233E/E240N T343R/N346D T175R/N178D 2 388 2903 2917 356R318Y/R338E/ R150Y/R170E/ 2 450 6645 6717 506 T343R/N346D/ T175R/N178D/R403E/E410N R233E/E240N R318Y/R338E/ R150Y/R170E/ 1 475 4989 5058 511Y345A/R403E/ Y177A/R233E/ E410N E240N R318Y/R338E/ R150Y/R170E/ 2 4926249 6347 607 Y345A/N346D/ Y177A/N178D/ R403E/E410N R233E/E240NY155F/K247N/ Y[155]F/K82N/ 2 622 10477 10973 791 N249S/R318Y/N84S/R150Y/ R338E/R403E R170E/R233E K247N/N249S/ K82N/N84S/ 2 805 80998569 814 R318Y/R338E/ R150Y/R170E/ R403E R233E Y155F/K247N/Y[155]F/K82N/ 2 618 9233 9709 801 N249S/R338E/ N84S/R170E/ R403E/E410NR233E/E240N R318Y/R338E/ R150Y/R170E/ 2 421 6107 6153 473 T343R/R403ET175R/R233E R318Y/R338E/ R150Y/R170E/ 2 529 793 815 391 T343R/E410NT175R/E240N R150Y/T343R/ R150Y/T175R/ 2 431 5020 5060 434 R403E/E410NR233E/E240N R170E/T343R/ R170E/T175R/ 2 484 5008 5060 450 R403E/E410NR233E/E240N Y155F/R338E/ Y[155]F/R170E/ 2 628 5406 5509 521 T343R/R403E/T175R/R233E/ E410N E240N Y155F/K247N/ K82N/N84S/ 2 513 9067 9267 642N249S/R318Y/ R150Y/R170E/ R338E/T343R/ T175R/R233E/ R403E/E410N E240NK247N/N249S/ K82N/N84S/ 2 536 8604 8824 672 R318Y/R338E/ R150Y/R170E/T343R/R403E/ T175R/R233E/ E410N E240N Y155F/K228N/ Y[155]F/K63N/ 2 7809033 9557 854 I251S/R318Y/ I86S/R150Y/ R338E/R403E/ R170E/R233E/ E410NE240N N260S/R318Y/ Y[155]F/N95S/ 2 539 8325 8537 675 R338E/T343R/R150Y/R170E/ R403E/E410N T175R/R233E/ E240N Y155F/N260S/ Y[155]F/N95S/ 1578 3266 6295 733 R318Y/R338E/ R150Y/R170E/ T343R/R403E/ T175R/R233E/E410N E240N K228N/K247N/ K63N/K82N/ 2 753 8972 9391 757 N249S/R318Y/N84S/R150Y/ R338E/T343R/ R170E/T175R/ R403E/E410N R233E/E240NY155F/R338E/ Y[155]F/R170E/ 2 503 5350 5412 506 T343R/R403E T175R/R233EY155F/R338E/ Y[155]F/R170E/ 2 589 5447 5546 526 T343R/R403E/T175R/R233E/ E410S E240S Y155F/N260S/ Y[155]F/N95S/R170E/ 2 485 95909749 619 R338E/T343R/ T175R/ R403E R233E Y155F/I251S/ Y[155]F/I86S/ 2732 7531 7926 807 R338E/T343R/ R170E/T175R/ R403E R233E R318Y/R338E/R150Y/R170E/ 2 618 4657 4728 466 T343R/R403E/ T175R/R233E/ E410S E240SY155F/K247N/ Y[155]F/K82N/ 2 866 7007 7391 751 N249S/T343R/ N84S/T175R/R403E R233E K247N/N249S/ K82N/N84S/ 2 804 9554 10051 776 R338E/T343R/R170E/T175R/ R403E/E410N R233E/E240N Y155F/K247N/ Y[155]F/K82N/ 2 6622965 3048 578 N249S/R318Y/ N84S/R150Y/ R338E R170E Y155F/K247N/Y[155]F/K82N/ 1 717 8404 8790 783 N249S/R338E/ N84S/R170E/ R403E R233EY155F/K247N/ Y[155]F/K82N/ 2 676 7455 7702 676 N249S/R338E/ N84S/R170E/T343R/R403E T175R/R233E K247N/N249S/ K82N/N84S/ 2 680 7758 8085 747T343R/R403E/ T175R/R233E/ E410N E240N Mutation Mutation (Mature FIX(Chymotrypsin Cmax/ Numbering) Numbering) Dose Vz Cl N157D N[157]D 9.9393 0.93 Y155F Y[155]F 18.1 149 0.23 A103N/N105S/ A[103]N/N[105]S/ 13.1325 0.40 Y155F Y[155]F D104N/K106S/ D[104]N/K[106]S/ 13.8 ± 1.02 243 ±47.4 0.341 ± 0.128 Y155F Y[155]F WT Catalyst 11.9 229 0.48 BiosciencesWT A103N/N105S A[103]N/N[105]S 12.5 177 0.33 D104N/K106S D[104]N/K[106]S13.9 164 0.26 K106N/V108S K[106]N/V[108]S 12.8 234 0.32  9.10 ± 0.518528 ± 184  0.619 ± 0.156 T148A BeneFIX, 9.12 ± 1.52 308 ± 160  0.662 ±0.071 T[148]A T148A T[148]A 10.2 ± 2.09 317 ± 82.3 0.582 ± 0.084 10.5251 0.64 D64N D[64]N 11.8 304 0.47 D64A D[64]A 11.5 359 0.68 N167DN[167]D 8.9 410 0.85 N167Q N[167]Q 8.20 ± 1.17 318 ± 42.5 0.655 ± 0.103S61A S[61]A 10.0 325 0.57 S53A S[53]A 11.2 238 0.43 T159A T[159]A 10.5315 0.97 T169A T[169]A 8.3 567 1.15 11.2 ± 2.89 295 ± 31.5 0.447 ± 0.13212.5 223 0.42 Y155H Y[155]H 11.6 253 0.38 Y155Q Y[155]Q 13.6 262 0.33S158E S[158]E 14.5 291 0.46 N157Q N[157]Q 11.3 395 0.83 9.43 ± 1.93 314± 91.3 0.449 ± 0.087 12.4 205 0.49 F205T D39N/F41T 12.3 ± 1.58 296 ±119  0.410 ± 0.118 14.4 255 0.30 K228N N[105]S/K63N D104N/K106S/D[104]N/ 13.6 513 0.45 K228N K[106]S/K63N Y155F/K228N Y[155]F/K63N 8.6431 0.47 D104N/K106S/ D[104]N/K[106]S/ 14.0 211 0.27 Y155F/K228NY[155]F/K63N I251S I86S 10.1 260 0.32 A103N/N105S/ A[103]N/ 14.3 2410.38 I251S N[105]S/I86S D104N/K106S/ D[104]N/ 13.9 375 0.38 I251SK[106]S/I86S Y155F/I251S Y[155]F/I86S 12.2 291 0.37 A262S A95bS 14.4 1880.36 E410N E240N 7.4 469 0.75 E239N E74N 10.7 257 0.54 10.8 ± 3.41 387 ±154  0.420 ± 0.106 10.6 420 0.53 N249S A103N/N105S/ A[103]N/N[105]S/21.5 226 0.21 K247N/N249S K82N/N84S D104N/K106S/ D[104]N/K[106]S/ 16.2426 0.51 K247N/N249S K82N/N84S D104N/K106S/ D[104]N/K[106]S/ 16.8 2330.27 Y155F/K247N/ Y[155]F/ N249S K82N/N84S S319N/L321S S151N/L153S 11.4210 0.42 11.5 ± 3.18 231 ± 156  0.295 ± 0.105 16.1 220 0.20 N260SK[106]S/N95S Y155F/N260S Y[155]F/N95S 18.4 257 0.27 Y284N Y117N 9.0 2700.46 R318Y/E410N R150Y/E240N 6.1 900 1.46 R338E/E410N R170E/E240N 6.2570 1.18 13.4 ± 2.03 196 ± 49.2 0.312 ± 0.063 E410N E240N 6.8 1080 1.25E410N E240N D203N/F205T/ D39N/F41T/ 9.3 281 0.63 R338E R170ED203N/F205T/ D39N/F41T/ 9.0 327 0.72 R338A R170A D203N/F205T/ D39N/F41T/8.7 732 1.91 R318Y R150Y D203N/F205T/ D39N/F41T/ 11.3 156 0.36R338E/R403E R170E/R233E K228N/E410N K63N/E240N 6.0 522 0.98 K228N/R338EK63N/R170E 10.4 207 0.58 R318Y/R338E/ R150Y/E240N/ 5.78 ± 1.56 778 ±272   1.62 ± 0.730 E410N R170E 8.9 355 0.49 R318Y/R338E/ R150Y/R170E/E410N E240N/ Y155F/R318Y/ Y[155]F/R150Y/ 6.5 1136 1.53 R338E/E410NR170E/E240N K228N/R318Y/ K63N/R150Y/ 6.0 485 1.10 E410N E240N 16.4 ±3.72 157 ± 38.3 0.246 ± 0.051 R403E/E410N R233E/E240N 16.2 157 0.26R318Y/R338E/ R150Y/R170E/ R403E/E410N R233E/E240N D104N/K106S/D[104]N/K[106]S/ 15.1 183 0.30 R318Y/R338E/ R150Y/R170E/ R403E/E410NR233E/E240N Y155F/R318Y/ Y[155]F/R150Y/ 12.4 226 0.27 R338E/R403E/R170E/R233E/ E410N E240N 17.2 ± 2.02 156 ± 8.74 0.167 ± 0.039Y155F/R318Y/ N[105]S/Y[155]F/ R338E/R403E/ R150Y/R170E/ E410NR233E/E240N 14.4 114 0.17 Y155F/R318Y/ K[106]S/Y[155]F/ R338E/R403E/R150Y/R170E/ E410N R233E/E240N D203N/F205T/ D39N/F41T/ 8.0 816 1.31R318Y/E410N R150Y/E240N R338L R170L 11.2 285 0.54 K316M K148M 7.9 3560.47 E239S E74S 11.3 278 0.41 E239A E74A 13.1 353 0.45 E239R E74R 8.9307 0.50 E239K E74K 14.4 278 0.48 H257F H92F 10.3 273 0.70 H257Y H92Y13.6 245 0.49 H257E H92E 10.9 294 0.42 H257S H92S 8.2 358 0.57 T412AT242A 7.1 379 0.58 T412V T242V 8.3 565 0.69 E410N/T412A E240N/T242A 2.81679 2.51 E410N/T412V E240N/T242V 3.7 906 2.27 E410Q E240Q 7.2 836 2.19E410S E240S 7.2 587 1.32 E410A E240A 8.6 946 1.81 E410D E240D 9.2 3200.63 N346D N178D 13.3 170 0.34 Y155F/N346D 178D/Y[155]F 17.0 176 0.26N346Y N178Y 11.7 365 0.77 Y345T Y177T 6.1 438 0.85 T343R Y175R 6.5 4870.85 T343E T175E 12.6 318 0.53 T343Q Y175Q 9.0 408 0.63 F342I F174I 8.3491 0.88 T343R/Y345T T175R/Y177T 9.1 422 0.90 R318Y/R338E R150Y/R170E11.2 336 0.69 K228N/I251S K63N/I86S 11.3 551 0.65 K228N/R318Y/K63N/R150Y/ 9.1 261 0.37 R338E/R403E/ R170E/R233E/ E410N E240NY155F/K228N/ Y[155]F/K63N/ 18.6 ± 2.14 158 ± 50.1 0.183 ± 0.062R318Y/R338E/ R150Y/R170E/ R403E/E410N R233E/E240N D85N/K228N/D[85]N/K63N/ 24.8 125 0.15 R318Y/R338E/ R150Y/R170E/ R403E/E410NR233E/E240N I251S/R318Y/ I86S/R150Y/ 15.7 ± 4.89 242 ± 89.4 0.438 ±0.171 R338E/R403E/ R170E/R233E/ E410N E240N D104N/K106S/D[104]N/K[106]S/ 17.2 ± 2.24 122 ± 10.1 0.123 ± 0.008 I251S/R318Y/I86S/R150Y/ R338E/R403E/ R170E/R233E/ E410N E240N 19.9 110 0.15R318Y/R338E/ R150Y/R170E/ R403E/E410N R233E/E240N I251S/R318Y/I86S/R150Y/ 7.5 664 0.78 R338E/E410N R170E/E240N D104N/K106S/D[104]N/K[106]S 16.4 650 0.62 I251S/R318Y/ I86S/R150Y/ R338E/E410NR170E/E240N K247N/N249S/ K82N/N84S/ 15.4 170 0.18 R318Y/R338E/R150Y/R170E/ R403E/E410N R233E/E240N Y155F/K247N/ Y[155]F/ 13.8 160 0.18N249S/R318Y/ K82N/N84S/ R338E/R403E/ R150Y/R170E/ E410N R233E/E240NA103N/N105S/ A[103]N/N[105]S 18.6 117 0.13 K247N/N249S/ K82N/N84S/R318Y/R338E/ R150Y/R170E/ R403E/E410N R233E/E240N/ D104N/K106S/D[104]N/K[106]S 19.4 140 0.15 K247N/N249S/ K82N/N84S/ R318Y/R338E/R150Y/R170E/ R403E/E410N R233E/E240N/ D104N/K106S/ D[104]N/K[106]S/ 18.4139 0.11 Y155F/K247N/ Y[155]F/K82N/ N249S/R318Y/ N84S/R150Y/R338E/R403E/ R170E/R233E/ E410N E240N K247N/N249S/ K82N/N84S/ 18.1 3960.54 R318Y/R338E/ R150Y/R170E/ E410N E240N Y155F/K247N/ Y[155]F/ 8.1 7450.83 N249S/R318Y/ K82N/N84S/ R338E/E410N R150Y/R170E/ E240N/R318Y/R338E/ R150Y/R170E/ 14.7 184 0.34 R403E/E410S R233E/E240SR318Y/R338E/ R150Y/R170E/ 7.7 1066 2.08 E410S E240S K228N/K247N/K63N/K82N/ 19.6 268 0.28 N249S N84S 18.5 ± 3.84 183 ± 63.3 0.160 ± 0.045Y155F/K228N/ Y[155]F/K63N/ K247N/N249S K82N/N84S 17.9 271 0.25K228N/K247N/ K63N/K82N/ N249S N84S Y155F/K228N/ Y[155]F/K63N/ 17.2 2460.31 K247N/N249S K82N/N84S 21.7 ± 3.19 96.6 ± 15.4  0.115 ± 0.030N249S/R318Y/ R150Y/R170E/ R338E/R403E/ R233E/E240N/ E410N D104N/K106S/D[104]N/K[106]S/ 24.4 ± 3.19 116 ± 15.4 0.100 ± 0.030 K228N/K247N/K63N/K82N/ N249S/R318Y/ N84S/R150Y/ R338E/R403E/ R170E/R233E/ E410NE240N 27.3 73 0.09 K247N/N249S/ K82N/N84S/ R318Y/R338E/ R150Y/R170E/R403E/E410N R233E/E240N R318Y/R338E/ R150Y/R170E/ 20.0 124 0.20R403E/E410N/ R233E/E240N/ T412V T242V R318Y/R338E/ R150Y/R170E/ 17.5 1070.20 R403E/E410N/ R233E/E240N/ T412A T242A R318Y/R338E/ R150Y/R170E/ 19.7 ± 0.684 144 ± 47.8 0.270 ± 0.145 R403E/T412A R233E/T242AR318Y/R338E/ R150Y/R170E/ 13.6 398 0.60 T412A T242A R318Y/R338E/150Y/R170E/ 16.3 772 2.31 E410N/T412V E240N/T242V N260S/R318Y/N95S/R150Y/ 23.9 111 0.13 R338E/R403E/ R170E/R233E/ E410N E240ND104N/K106S/ D[104]N/K[106]S/ 17.2 145 0.12 N260S/R318Y/ N95S/R150Y/R338E/R403E/ R170E/R233E/ E410N E240N Y155F/N260S/ Y[155]F/N95S/ 21.4167 0.21 R318Y/R338E/ R150Y/R170E/ R403E/E410N R233E/E240N R318Y/R338E/R150Y/R170E/ 25.2 86 0.12 N346D/R403E/ N178D/R233E/ E410N E240NY155F/R318Y/ Y[155]F/R150Y/ 18.2 154 0.20 R338E/N346D/ R170E/N178D/R403E/E410N R233E/E240N K247N/N249S/ K82N/N84S/ 17.4 186 0.23 N260S N95SY155F/K247N/ Y[155]F/K82N/ 24.0 244 0.22 N249S/N260S N84S/N95SD104N/K106S/ D[104]N/K[106]S/ 16.5 235 0.23 K247N/N249S/ K82N/N84S/N260S N95S D104N/K106S/ D[104]N/K[106]S/ 23.3 134 0.14 Y155F/K247N/Y[155]F/K82N N249S/N260S N84S/N95S/ K247N/N249S/ K82N/N84S/ 18.9 1490.13 N260S/R318Y/ N95S/R150Y/ R338E/R403E/ R170E/R233E/ E410N E240NY155F/K247N/ Y[155]F/K82N/ 21.0 160 0.11 N249S/N260S/ N84S/N95S/R318Y/R338E/ R150Y/R170E/ R403E/E410N R233E/E240N R318Y/R338E/T175R/R233E/ 20.5 200 0.27 T343R/R403E/ E240N/R150Y/ E410N R170EY155F/R318Y/ Y[155]F/R150Y/ 20.5 61 0.23 R338E/T343R/ R170E/T175R/R403E/E410N R233E/E240N D104N/K106S/ D[104]N/K[106]S/ 21.7 ± 4.71 128 ±21.1 0.133 ± 0.013 R318Y/R338E/ R150Y/R170E/ T343R/R403E/ T175R/R233E/E410N E240N R338E/T343R R170E/T175R 12.8 949 1.23 T343R/N346YT175R/N178Y 12.3 ± 5.14 394 ± 156  0.989 ± 0.360 R318Y/R338E/R150Y/R170E/ 24.7 189 0.40 N346Y/R403E/ N178Y/R233E/ E410N E240N 22.2118 0.27 T343R/N346Y/ T175R/N178Y/ R403E/E410N R233E/E240N T343R/N346DT175R/N178D 17.0 192 0.34 R318Y/R338E/ R150Y/R170E/ 20.7 97 0.15T343R/N346D/ T175R/N178D/ R403E/E410N R233E/E240N R318Y/R338E/R150Y/R170E/ 22.3 135 0.20 Y345A/R403E/ Y177A/R233E/ E410N E240NR318Y/R338E/ R150Y/R170E/ 22.1 112 0.16 Y345A/N346D/ Y177A/N178D/R403E/E410N R233E/E240N Y155F/K247N/ Y[155]F/K82N/ 26.9 85 0.10N249S/R318Y/ N84S/R150Y/ R338E/R403E R170E/R233E K247N/N249S/ K82N/N84S/20.0 137 0.12 R318Y/R338E/ R150Y/R170E/ R403E R233E Y155F/K247N/Y[155]F/K82N/ 22.4 92 0.10 N249S/R338E/ N84S/R170E/ R403E/E410NR233E/E240N R318Y/R338E/ R150Y/R170E/ 19.9 99 0.16 T343R/R403ET175R/R233E R318Y/R338E/ R150Y/R170E/ 5.6 931 1.23 T343R/E410NT175R/E240N R150Y/T343R/ R150Y/T175R/ 20.7 130 0.21 R403E/E410NR233E/E240N R170E/T343R/ R170E/T175R/ 19.8 141 0.20 R403E/E410NR233E/E240N Y155F/R338E/ Y[155]F/R170E/ 17.9 164 0.18 T343R/R403E/T175R/R233E/ E410N E240N Y155F/K247N/ K82N/N84S/ 24.7 82 0.11N249S/R318Y/ R150Y/R170E/ R338E/T343R/ T175R/R233E/ R403E/E410N E240NK247N/N249S/ K82N/N84S/ 24.4 89 0.12 R318Y/R338E/ R150Y/R170E/T343R/R403E/ T175R/R233E/ E410N E240N Y155F/K228N/ Y[155]F/K63N/ 20.5123 0.11 I251S/R318Y/ I86S/R150Y/ R338E/R403E/ R170E/R233E/ E410N E240NN260S/R318Y/ Y[155]F/N95S/ 24.0 92 0.12 R338E/T343R/ R150Y/R170E/R403E/E410N T175R/R233E/ E240N Y155F/N260S/ Y[155]F/N95S/ 20.4 133 0.16R318Y/R338E/ R150Y/R170E/ T343R/R403E/ T175R/R233E/ E410N E240NK228N/K247N/ K63N/K82N/ 26.0 117 0.11 N249S/R318Y/ N84S/R150Y/R338E/T343R/ R170E/T175R/ R403E/E410N R233E/E240N Y155F/R338E/Y[155]F/R170E/ 16.7 135 0.19 T343R/R403E T175R/R233E Y155F/R338E/Y[155]F/R170E/ 22.9 156 0.18 T343R/R403E/ T175R/R233E/ E410S E240SY155F/N260S/ Y[155]F/N95S/R170E/ 24.0 74 0.10 R338E/T343R/ T175R/ R403ER233E Y155F/I251S/ Y[155]F/I86S/ 21.0 134 0.13 R338E/T343R/ R170E/T175R/R403E R233E R318Y/R338E/ R150Y/R170E/ 19.9 199 0.23 T343R/R403E/T175R/R233E/ E410S E240S Y155F/K247N/ Y[155]F/K82N/ 18.3 169 0.14N249S/T343R/ N84S/T175R/ R403E R233E K247N/N249S/ K82N/N84S/ 20.4 1160.10 R338E/T343R/ R170E/T175R/ R403E/E410N R233E/E240N Y155F/K247N/Y[155]F/K82N/ 13.6 313 0.33 N249S/R318Y/ N84S/R150Y/ R338E R170EY155F/K247N/ Y[155]F/K82N/ 16.9 118 0.11 N249S/R338E/ N84S/R170E/ R403ER233E Y155F/K247N/ Y[155]F/K82N/ 20.3 131 0.13 N249S/R338E/ N84S/R170E/T343R/R403E T175R/R233E K247N/N249S/ K82N/N84S/ 18.0 122 0.13T343R/R403E/ T175R/R233E/ E410N E240N

Example 7 In Vivo Assessment of FIX Polypeptide Procoagulant Activity

Mouse models of hemophilia B, using mice deficient in FIX (FIX^(−/−)mice), were established to assess the procoagulant activity of FIXpolypeptides. The mice were treated with FIX polypeptide and the amountof blood lost in 20 minutes was measured to determine the procoagulantactivity of the FIX polypeptides.

A. In Vivo Assessment of Wild-Type FIX Procoagulant Activity

Male FIX^(−/−) mice were anesthetized by intraperitoneal administrationof a ketamine/xylazine cocktail (45 mg/ml and 3.6 mg/ml in saline) andplaced on a heated platform (39° C.) to ensure there was no drop in bodytemperature. The procedure room was kept at a temperature of 82° F. Tenminutes prior to tail cut the tail was immersed in 10 mL of pre-warmedPBS (15 mL centrifuge tube; 39° C.). Seven to fifteen mice were injectedwith recombinant human FIX (Benefix® Coagulation Factor IX(Recombinant), Wyeth) or modified FIX polypeptides diluted in a bufferthat was the same as that of Benefix® Coagulation Factor IX(Recombinant) (0.234% sodium chloride, 8 mM L-histidine, 0.8% sucrose,208 mM glycine, 0.004% polysorbate 80) via the tail vein in a singleinjection. A negative control group of mice received buffer only. Ininstances where the injection was missed, the animal was excluded fromthe study.

Injection with FIX polypeptide or buffer was made 5 minutes prior totail cut. The tail cut was made using a razor blade 5 mm from the end ofthe tail and blood was collected into PBS for a period of 20 minutes. Atthe end of the collection period, total blood loss was assessed. Thecollection tubes were mixed and a 1 ml aliquot of each sample was takenand assayed for hemoglobin content. Triton X-100 was diluted 1 in 4 insterile water and 100 μL was added to the 1 mL samples to causehemolysis. The absorbance of the samples was then measured at awavelength of 546 nm. To calculate the amount of blood lost, theabsorbance was read against a standard curve generated by measuring theabsorbance at 546 nm of known volumes of murine blood, diluted in PBSand hemolyzed as above with Triton X 100. Values are expressed asMean±SEM.

1. Dose Response Study Assessing Wild-Type FIX Coagulant Activity

Dose response studies to assess the coagulant activity of Benefix®Coagulation Factor IX (Recombinant) at 0.03, 0.1, 0.3 and 1 mg/kg inFIX^(−/−) mice were performed. In this experiment, the blood loss in thebuffer-only group was 835.42±24.55 μL which was significantly reduced byBenefix® Coagulation Factor IX (Recombinant) treatment at 0.1, 0.3 and 1mg/kg (to 558.59±56.63 μL, 415.81±66.72 μL and 270.75±57.48 μL; p<0.05using Kruskal-Wallis followed by Dunn's post test). At the lowest dosetested of 0.03 mg/kg the value was 731.66±59.16 μL. Calculated ED₅₀values using non-linear regression are shown in Table 28 below.

2. Dose Response Assessing the Coagulant Activity ofFIXa-R318Y/R338E/R403E/E410N, FIXa-R318Y/R338E/E410N andFIXa-Y155F/K247N/N249S/R318Y/R338E/R403E/E410N

Dose response studies were conducted in which the coagulant activity ofFIXa-R318Y/R338E/R403E/E410N (R150Y/R170E/R233E/E240N by chymotrypsinnumbering), FIXa-R318Y/R338E/E410N (R150Y/R170E/E240N by chymotrypsinnumbering) and FIXa-Y155F/K247N/N249S/R318Y/R338E/R403E/E410N(Y[155]F/K82N/N84S/R150Y/R170E/R233E/E240N by chymotrypsin numbering) atdifferent doses were assessed.

Treatment with FIXa-R318Y/R338E/R403E/E410N resulted in significantinhibition of blood loss at 0.01, 0.03, 0.1, 0.3 and 1 mg/kg(434.65±73.75 μL, 497.28±50.92 μL, 230.81±39.67 μL, 261.94±58.79 μL and251.56±41.81 μL, respectively) compared to the buffer-only control(811.45±26.63 μL; p<0.05 using Kruskal-Wallis followed by Dunn's posttest). Reducing the dose to 0.003 mg/kg led to blood loss values nearercontrol levels, of 786.83±44.39 μL.

Treatment with FIXa-R318Y/R338E/E410N also resulted in significantinhibition of blood loss at 0.03, 0.1, 0.3 and 1 mg/kg (571.67±50.45 μL,425.42±43.65 μL, 263.47±42.66 μL and 78.19±13.42 μL, respectively)compared to the buffer-only control (845.14±23.63 μL; p<0.05 usingKruskal-Wallis followed by Dunn's post test). Reducing the dose to 0.001mg/kg led to blood loss values nearer control levels, of 777.16±53.72μL.

Treatment with FIXa-Y155F/K247N/N249S/R318Y/R338E/R403E/E410N resultedin the most significant inhibition of blood loss of the mutants tested:460.03±74.60 μL, 393.48±75.16 μL and 157.28±28.89 μL at 0.01, 0.03 and0.1 mg/kg, respectively, compared to the buffer-only control(851.38±44.25 μL; p<0.05 using Kruskal-Wallis followed by Dunn's posttest). Calculated ED₅₀ values using non-linear regression are shown inTable 28 below.

TABLE 28 Blood Loss; Mutation (Mature FIX Mutation (chymotrypsin n/ nED50 numbering) numbering group (expts) (mg/kg) BeneFIX Benefix ®Coagulation BeneFIX Benefix ®  7-20 2 0.2 FIX (T148A) Coagulation FIX(T[148]A) R318Y/R338E/R403E/E410N R150Y/R170E/R233E/E240N 19-38 3 0.02R318Y/R338E/E410N R150Y/R170E/E240N  8-42 4 0.06Y155F/K247N/N249S/R318Y/ Y[155]F/K82N/N84S/R150Y/ 18-21 2 0.01R338E/R403E/E410N R170E/R233E/E240N

3. Duration Response Assessing Wild-Type FIX Coagulant Activity

Studies were performed to assess the duration of effect of Benefix®Coagulation Factor IX (Recombinant) at 0.5 mg/kg in FIX^(−/−) mice. Micewere dosed intravenously at 48 hr, 24 hr, 16 hr, 8 hr, 4 hr, 2 hr, 30min and 5 min prior to tail cut. In this experiment, inhibition from thecontrol group was determined where the control group was set at 0%inhibition. Inhibition of blood loss was 59.7±11.9%, 48.25±12.84%,57.74±9.10%, 56.04±8.46%, 32.09±7.92%, 12.94±7.33%, 38.75±11.47% and0.64±11.3% at 5 min, 30 min, 2, 4, 8, 16, 24 and 48 hr, respectivelyfrom vehicle control (Mean and SEM, n=8-33 mice, from 3 experiments).

4. Duration Response Assessing FIXa-R318Y/R338E/R403E/E410N CoagulantActivity

Studies were performed to assess the duration of effect ofFIXa-R318Y/R338E/R403E/E410N at 0.5 mg/kg in FIX^(−/−) mice. Mice weredosed i.v. at 96 hr, 72 hr, 48 hr, 32 hr, 24 hr, 16 hr, 8 hr, 4 hr, 2hr, 30 min and 5 min prior to tail cut. In this experiment, inhibitionfrom the control group was determined where the control group was set at0% inhibition. Inhibition of blood loss was 93.26±2.04%, 96.30±3.70%,85.86±6.52%, 69.4±9.92%, 89.05±3.69%, 78.48±8.71%, 63.33±6.70%,47.97±10.07%, 3.1±8.22%, −13.52±10.59% and −12.82±7.31% at 5 min, 30min, 2, 4, 8, 16, 24, 32, 48, 72 and 96 hr, respectively from vehiclecontrol (Mean and SEM, n=8-45 mice, from 4 experiments).

Note on the FIX^(−/−) Mice:

The FIX knockout colony of mice was generated by in vitro fertilizationusing cryo-preserved sperm from male FIX knock out mice. All offspringwere genotyped using PCR-based protocols to select those animals thatcontained a FIX knock-out allele. Further crossings of these animals andtheir offspring (after PCR-based genotyping) produced FIX knock-outanimals (i.e., hemizygous males and homozygous females because the FIXgene is on the X chromosome), as confirmed by PCR. After PCRconfirmation of the genotype of all members of this initial FIX colony,PCR confirmation of all colony offspring was ceased since legitimateknock-out animals can only produce knock-out offspring. “Retiredbreeders” from the colony were, however, genotyped on several occasions.Approximately 7 months after genotyping of all colony offspring wasceased, genotyping of retired breeders clearly indicated the presence ofnon-knock-out (or wild-type) animals in the colony. Based on thisresult, all members of the knock-out colony were genotyped and anynon-knock-out animals were identified and eliminated from the colony.The results of the colony genotyping indicated that 19% of the male micewere wild type and 4% of the male animals were ambiguous due to poor DNApreparations. Both the wild type and “ambiguous” males (and females)were eliminated from the colony.

Thus, the FIX knockout colony was contaminated at some point with one ormore non-knock-out animals and therefore contained a small fraction ofnon-knock out animals that increased over time until between 19-23% ofthe males in the colony contained a wild type FIX gene (in vivoexperiments use male mice only). With respect to the FIX data generatedand reported in this application, all of the in vitro data isunaffected. With respect to in vivo data, it is assumed and expectedthat the contamination affected all compounds similarly and thereforedoes not affect either the rank order of variants or their comparison toBeneFIX. Since the contaminating animals already had endogenous FIX,they would lose much less blood in the efficacy and duration experimentsthan true hemophilic animals and would benefit much less fromadministration of exogenous FIX, therefore increasing the “spread” orvariability of data for all compounds. The contamination also could makeall the compounds appear slightly less potent than they actually are,but their ratio to BeneFIX should not be altered (i.e., the potency andduration advantage of our lead molecules should be unaffected).

B. In Vivo Assessment of Wild-Type FIX Procoagulant Activity—New ColonyData

The data described below comes from a new colony, rebuilt from theconfirmed FIX−/− mice described above. Mice were double confirmed bygenotyping before being used as breeders. All data described below comesfrom mice born from breeding units where parents have been doubleconfirmed. All replacement breeders are also double confirmed as FIX−/−prior to initiation of new breeding units.

Male FIX^(−/−) mice were anesthetized by intraperitoneal administrationof a ketamine/xylazine cocktail (45 mg/ml and 3.6 mg/ml in saline) andplaced on a heated platform (39° C.) to ensure there was no drop in bodytemperature. The procedure room was kept at a temperature of 82° F. Tenminutes prior to tail cut the tail was immersed in 10 mL of pre-warmedPBS (15 mL centrifuge tube; 39° C.). Seven to fifteen mice were injectedwith recombinant human FIX (Benefix® Coagulation Factor IX(Recombinant), Wyeth) or modified FIX polypeptides diluted in a bufferthat was the same as that of Benefix® Coagulation Factor IX(Recombinant) (0.234% sodium chloride, 8 mM L-histidine, 0.8% sucrose,208 mM glycine, 0.004% polysorbate 80) via the tail vein in a singleinjection. A negative control group of mice received buffer only. Ininstances where the injection was missed, the animal was excluded fromthe study.

Injection with FIX polypeptide or buffer was made 5 minutes prior totail cut. The tail cut was made using a razor blade 5 mm from the end ofthe tail and blood was collected into PBS for a period of 20 minutes. Atthe end of the collection period, total blood loss was assessed. Thecollection tubes were mixed and a 1 ml aliquot of each sample was takenand assayed for hemoglobin content. Triton X-100 was diluted 1 in 4 insterile water and 100 μL was added to the 1 mL samples to causehemolysis. The absorbance of the samples was then measured at awavelength of 546 nm. To calculate the amount of blood lost, theabsorbance was read against a standard curve generated by measuring theabsorbance at 546 nm of known volumes of murine blood, diluted in PBSand hemolyzed as above with Triton X 100. Values are expressed asMean±SEM.

1. Dose Response Studies Assessing FIX Coagulant Activity

Dose response studies to assess the coagulant activity of Benefix®Coagulation Factor IX (Recombinant) and FIX polypeptides at varyingdoses in FIX^(−/−) mice were performed. In these experiments ED₅₀ valueswere calculated using non-linear regression and are shown in Table 29below.

TABLE 29 Dose Response ED₅₀ values Average Mutation (Chymotrypsinn/group/ N ED50 Mutation numbering) expt (expts) (mg/kg) BeneFIX BeneFIX10-14 2 0.4 WT Catalyst Biosciences WT  8-15 4 1.6 T148A T[148]A 10-15 21.0 R318Y/R338E/E410N R150Y/R170E/E240N 10-13 2 0.14 R318Y/R403E/E410NR150Y/R233E/E240N 13-15 2 0.095 R318Y/R338E/R403E/E410NR150Y/R170E/R233E/E240N  7-14 6 0.02 D104N/K106S/Y155F/R318Y/D[104]N/K[106]S/Y[155]F/  9-14 4 0.05 R338E/R403E/E410NR150Y/R170E/R233E/E240N T343R T175R  9-15 4 0.9 Y155F/K228N/R318Y/R338E/Y[155]F/K63N/R150Y/R170E/ 10-14 2 0.08 R403E/E410N R233E/E240NI251S/R318Y/R338E/E410N I86S/R150Y/R170E/E240N  9-18 3 1.0K247N/N249S/R318Y/R338E/ K82N/N84S/R150Y/R170E/  9-14 4 0.06 R403E/E410NR233E/E240N Y155F/K247N/N249S/R318Y/ Y[155]F/K82N/N84S/R150Y/  9-15 40.03 R338E/R403E/E410N R170E/R233E/E240N A103N/N105S/K247N/N249S/A[103]N/N[105]S/K82N/N84S/  8-10 2 0.08 R318Y/R338E/R403E/E410NR150Y/R170E/R233E/E240N D104N/K106S/Y155F/K247N/D[104]N/K[106]S/Y[155]F/ 12-15 2 0.055 N249S/R318Y/R338E/R403E/K82N/N84S/R150Y/R170E/ E410N R233E/E240N R318Y/R338E/R403E/E410SR150Y/R170E/R233E/E240S 10-15 2 0.055 D104N/K106S/Y155F/K228N/D[104]N/K[106]S/Y[155]F/ 10-12 1 1.64 K247N/N249S K63N/K82N/N84SK228N/K247N/N249S/R318Y/ K63N/K82N/N84S/R150Y/  8-15 5 0.08R338E/R403E/E410N R170E/R233E/E240N D104N/K106S/K228N/K247N/D[104]N/K[106]S/K63N/K82N/ 13-15 2 0.125 N249S/R318Y/R338E/R403E/N84S/R150Y/R170E/R233E/ E410N E240N Y155F/K228N/K247N/N249S/Y[155]F/K63N/K82N/N84S/ 12-15 2 0.035 R318Y/R338E/R403E/E410NR150Y/R170E/R233E/E240N R318Y/R338E/R403E/E410N/R150Y/R170E/R233E/E240N/  8-14 3 0.03 T412V T242VR318Y/R338E/R403E/E410N/ R150Y/R170E/R233E/E240N/ 11-15 2 0.04 T412AT242A K247N/N249S/N260S/R318Y/ K82N/N84S/N95S/R150Y/  8-15 4 0.26R338E/R403E/E410N R170E/R233E/E240N Y155F/K247N/N249S/N260S/Y[155]F/K82N/N84S/N95S/ 13-15 3 0.06 R318Y/R338E/R403E/E410NR150Y/R170E/R233E/E240N R318Y/R338E/T343R/R403E/R150Y/R170E/T175R/R233E/  7-15 5 0.025 E410N E240NY155F/R318Y/R338E/T343R/ Y[155]F/R150Y/R170E/T175R/ 10-14 2 0.0045R403E/E410N R233E/E240N D104N/K106S/R318Y/R338E/D[104]N/K[106]S/R150Y/R170E/ 10-15 3 0.07 T343R/R403E/E410NT175R/R233E/E240N R338E/T343R R170E/T175R 11-14 2 0.83R318Y/R338E/T343R/N346Y/ R150Y/R170E/T175R/N178Y/  9-13 3 0.03R403E/E410N R233E/E240N Y155F/K247N/N249S/R318Y/Y[155]F/K82N/N84S/R150Y/ 11-15 2 0.145 R338E/R403E R170E/R233EY155F/K247N/N249S/R338E/ Y[155]F/K82N/N84S/R170E/ 12-15 3 0.08R403E/E410N R233E/E240N R318Y/R338E/T343R/R403E R150Y/R170E/T175R/R233E10-15 2 0.025 Y155F/R318Y/R338E/T343R/ Y[155]F/R150Y/R170E/T175R/ 10-142 0.007 R403E R233E R318Y/R338E/T343R/E410N R150Y/R170E/T175R/E240N11-15 5 0.13 R318Y/T343R/R403E/E410N R150Y/T175R/R233E/E240N 10-15 20.03 Y155F/R318Y/T343R/R403E/ Y[155]F/R150Y/T175R/R233E/ 13-15 2 0.07E410N E240N R338E/T343R/R403E/E410N R170E/T175R/R233E/E240N 11-15 20.045 Y155F/R338E/T343R/R403E/E410N Y[155]F/R170E/T175R/R233E/ 10-15 20.055 E240N Y155F/K247N/N249S/R318Y/ Y[155]F/K82N/N84S/R150Y/ 11-15 20.04 R338E/T343R/R403E/E410N R170E/T175R/R233E/E240NK247N/N249S/R318Y/R338E/ K82N/N84S/R150Y/R170E/ 11-15 2 0.035T343R/R403E/E410N T175R/R233E/E240N K228N/I251S/R318Y/R338E/K63N/I86S/R150Y/R170E/ 10-15 3 0.01 R403E/E410N R233E/E240NY155F/K228N/I251S/R318Y/ Y[155]F/K63N/I86S/R150Y/ 13-15 2 0.04R338E/R403E/E410N R170E/R233E/E240N N260S/R318Y/R338E/T343R/N95S/R150Y/R170E/T175R/ 12-15 2 0.03 R403E/E410N R233E/E240NY155F/N260S/R318Y/R338E/ Y[155]F/N95S/R150Y/R170E/ 10-15 2 0.02T343R/R403E/E410N T175R/R233E/E240N K228N/K247N/N249S/R318Y/K63N/K82N/N84S/R150Y/ 12-15 3 0.03 R338E/T343R/R403E/E410NR170E/T175R/R233E/E240N Y155F/R338E/T343R/R403EY[155]F/R170E/T175R/R233E 12-15 1 0.06 R338E/T343R/R403ER170E/T175R/R233E 10-15 2 0.195 Y155F/R338E/T343R/R403E/Y[155]F/R170E/T175R/R233E/ 12-15 3 0.06 E410S E240SY155F/N260S/R338E/T343R/ Y[155]F/N95S/R170E/T175R/ 12-15 1 0.1 R403ER233E Y155F/I251S/R338E/T343R/ Y[155]F/I86S/R170E/T175R/ 13-15 2 0.145R403E R233E R318Y/R338E/T343R/R403E/ R150Y/R170E/T175R/R233E/ 11-15 20.015 E410S E240S Y155F/K247N/N249S/T343R/ Y[155]F/K82N/N84S/T175R/12-14 2 0.26 R403E R233E Y155F/K247N/N249S/R318Y/Y[155]F/K82N/N84S/R150Y/ 10-14 2 0.006 R338E/T343R/R403ER170E/T175R/R233E K247N/N249S/R318Y/R338E/ K82N/N84S/R150Y/R170E/ 10-132 0.009 T343R/R403E T175R/R233E Y155F/K247N/N249S/R338E/Y[155]F/K82N/N84S/R170E/ 12-13 1 0.2 T343R/R403E/E410N T175R/R233E/E240NK247N/N249S/R338E/T343R/ K82N/N84S/R170E/T175R/ 11-14 1 0.01 R403E/E410NR233E/E240N Y155F/K247N/N249S/R318Y/ Y[155]F/K82N/N84S/R150Y/ 13-15 20.04 R338E R170E K247N/N249S/R338E/T343R/ K82N/N84S/R170E/T175R/ 10-15 20.18 R403E/E410N R233E/E240N Y155F/K247N/N249S/R338E/Y[155]F/K82N/N84S/R170E/ 12-15 2 0.22 R403E R233EY155F/K247N/N249S/R318Y/ Y[155]F/K82N/N84S/R150Y/ 12-15 2 0.12R338E/T343R/E410N R170E/T175R/E240N K247N/N249S/R318Y/R338E/K82N/N84S/R150Y/R170E/ 11-14 2 0.12 T343R/E410N T175R/E240NY155F/K247N/N249S/R318Y/ Y[155]F/K82N/N84S/R150Y/ 10-15 2 0.07T343R/R403E/E410N T175R/R233E/E240N K247N/N249S/R318Y/T343R/K82N/N84S/R150Y/T175R/ 14-15 1 0.02 R403E/E410N R233E/E240NY155F/K247N/N249S/R318Y/ Y[155]F/K82N/N84S/R150Y/ 11-14 2 0.065T343R/R403E T175R/R233E Y155F/K247N/N249S/R318Y/Y[155]F/K82N/N84S/R150Y/ 12-15 1 0.25 T343R/E410N T175R/E240NY155F/K247N/N249S/R338E/ Y[155]F/K82N/N84S/R170E/ 10-15 2 0.125T343R/R403E T175R/R233E Y155F/K247N/N249S/T343R/Y[155]F/K82N/N84S/T175R/ 13-14 1 0.1 R403E/E410N R233E/E240NY155F/K247N/N249S/R318Y/ Y[155]F/K82N/N84S/R150Y/ 13-14 2 0.07R338E/T343R R170E/T175R Y155F/K247N/N249S/T343R/Y[155]F/K82N/N84S/T175R/ 11-15 1 0.11 E410N E240N

2. Duration Response Assessing Wild-Type FIX Coagulant Activity

Studies were performed to assess the duration of effect of Benefix®Coagulation Factor IX (Recombinant) at 0.5 mg/kg in FIX^(−/−) mice. Micewere dosed intravenously at 48 hr, 32 hr, 24 hr, 16 hr, 8 hr, 4 hr, 2 hrand 5 min prior to tail cut. In this experiment, inhibition from thecontrol group was determined where the control group was set at 0%inhibition Inhibition of blood loss was 68.6±5.8%, 64±6.98%, 54.7±6.13%,43.4±6.86%, 13.7±5.53%, 24.9±6.11%, 11.7±4.88% and 5.6±4.17% at 5 min,2, 4, 8, 16, 24, 32 and 48 hr, respectively from vehicle control (Meanand SEM, n=10-35 mice, from 3 experiments).

3. Duration Response Assessing FIX Polypeptide Procoagulant Activity

Studies were performed to assess the duration of effect ofFIX-polypeptides at 0.5 mg/kg in FIX^(−/−) mice. Mice were dosed i.v. at72 hr, 48 hr, 32 hr, 24 hr, 8 hr and 5 min prior to tail cut, or at 72hr, 48 hr and 1 hr prior to tail cut. In these experiments, inhibitionfrom the control group was determined where the control group was set at0% inhibition Inhibition of blood loss is shown as % inhibition (Meanand SEM) in Table 30.

TABLE 30 Inhibition of blood loss Mutation (chymotrypsin n/ N Inhibition(% of vehicle (0) +/− SEM) at each time point (hrs) numbering) group(expt) 0.08 1 8 24 R150Y/R170E/R233E 24-30 2   85 +/− 3.2 88.8 +/− 2.859.5 +/− 7.3 R150Y/R170E/E240N 37-44 3 71.6 +/− 3.9 85.0 +/− 3.8 59.4+/− 6.8 Y[155]F/R150Y/R170E/E240N 26-29 2 74.2 +/− 6.5 R150Y/R233E/E240N23-29 2 71.0 +/− 3.7 71.4 +/− 6.6 31.1 +/− 6.1 R150Y/R170E/R233E/E240N75-86 7 75.9 +/− 2 82.7 +/− 2.6   58 +/− 4.8 Y[155]F/R150Y/R170E/R233E/25-30 2 88.5 +/− 1.7 E240N D[104]N/K[106]S/Y[155]F/ 35-44 3 70.8 +/− 3.085.5 +/− 3.5 55.1 +/− 5.4 R150Y/R170E/R233E/E240N T175R 23-28 2 43.7 +/−6.3 30.9 +/− 6.6 23.8 +/− 3.8 Y[155]F/K63N/R150Y/R170E/ 36-43 3 65.2 +/−3.0 72.2 +/− 4.5 59.2 +/− 6.5 R233E/E240N K82N/N84S/R150Y/R170E/ 37-41 378.7 +/− 2.5 85.9 +/− 2.6 52.5 +/− 5.5 R233E/E240NY[155]F/K82N/N84S/R150Y/ 57-65 5 79.1 +/− 2.2 79.5 +/− 2.7 66.7 +/− 4.0R170E/R233E/E240N D[104]N/K[106]S/Y[155]F/K82N/ 20-29 2 71.2 +/− 4.574.2 +/− 6.6 61.2 +/− 7.2 N84S/R150Y/R170E/R233E/ E240NK82N/N84S/R150Y/R170E/ 23-28 2 76.0 +/− 6.6 E240NY[155]F/K82N/N84S/R150Y/ 26-30 2 77.7 +/− 5.1 R170E/E240NR150Y/R170E/R233E/E240S 35-42 3 79.3 +/− 1.9 75.6 +/− 4.6 51.0 +/− 5.4K63N/K82N/N84S/R150Y/ 32-38 3 72.6 +/− 2.9 78.6 +/− 3.7 44.2 +/− 7R170E/R233E/E240N D[104]N/K[106]S/K63N/K82N/ 26-28 2 81.6 +/− 3.5 86.0+/− 3.6 46.8 +/− 8.0 N84S/R150Y/R170E/R233E/ E240NY[155]F/K63N/K82N/N84S/ 23-29 2 85.5 +/− 2.2 75.6 +/− 4.0 70.6 +/− 6.5R150Y/R170E/R233E/E240N R150Y/R170E/R233E/E240N/ 40-44 3 69.5 +/− 3.285.5 +/− 2.6 37.5 +/− 5.1 T242V R150Y/R170E/R233E/E240N/ 29-38 3 81.3+/− 2.5 85.6 +/− 3.3 45.2 +/− 6.2 T242A K82N/N84S/N95S/R150Y/R170E/20-28 2 46.4 +/− 6.6 37.7 +/− 7.5   4.0 +/− 2.6 R233E/E240NY[155]F/K82N/N84S/N95S/ 37-43 3 72.2 +/− 4.4 69.1 +/− 5.4 47.0 +/− 6.1R150Y/R170E/R233E/E240N R150Y/R170E/T175R/R233E/ 32-38 3 80.3 +/− 2.678.2 +/− 3.8 68.3 +/− 5.5 E240N Y[155]F/R150Y/R170E/T175R/ 21-27 2 84.8+/− 2.5 87.8 +/− 2.8 76.6 +/− 4.2 R233E/E240ND[104]N/K[106]S/R150Y/R170E/ 26-30 2 80.4 +/− 2.8 81.5 +/− 4.8 69.5 +/−7.6 T175R/R233E/E240N R150Y/R170E/T175R/N178Y/ 35-43 3 76.6 +/− 3.1 85.1+/− 3.3 43.9 +/− 5.7 R233E/E240N Y[155]F/K82N/N84S/R150Y/ 24-30 2 76.2+/− 3.0 85.6 +/− 4.7 49.6 +/− 6.5 R170E/R233E K82N/N84S/R150Y/R170E/27-29 2 70.0 +/− 5.8 R233E Y[155]F/K82N/N84S/R170E/ 38-44 3 69.8 +/− 4.778.4 +/− 4.1 56.4 +/− 5.9 R233E/E240N K82N/N84S/R170E/R233E/ 28-30 263.9 +/− 7.2 E240N R150Y/R170E/T175R/R233E 37-43 3 80.0 +/− 2.1 83.5 +/−3.5 62.1 +/− 5.6 Y[155]F/R150Y/R170E/T175R/ 24-28 2 80.4 +/− 3.0 90.7+/− 2.1 65.7 +/− 6.6 R233E R150Y/R170E/T175R/E240N 35-44 3 65.5 +/− 4.774.1 +/− 5.3 55.8 +/− 5.6 R150Y/T175R/R233E/E240N 29-30 2 74.1 +/− 3.677.7 +/− 3.9 55.3 +/− 7.5 Y[155]F/R150Y/T175R/R233E/ 25-29 2 92.7 +/−2.1 E240N R170E/T175R/R233E/E240N 26-30 2   67 +/− 5.3 87.4 +/− 4.2 55.9+/− 8.7 Y[155]F/R170E/T175R/R233E/ 34-43 3 77.8 +/− 4.2 90.8 +/− 2.868.6 +/− 5.2 E240N Y[155]F/K82N/N84S/R150Y/ 39-43 3 76.0 +/− 3.0 80.4+/− 3.3 72.7 +/− 3.8 R170E/T175R/R233E/E240N K82N/N84S/R150Y/R170E/42-44 3 83.0 +/− 2.4 81.0 +/− 2.4 73.8 +/− 5.2 T175R/R233E/E240NK63N/I86S/R150Y/R170E/R233E/ 21-26 2 71.9 +/− 3.6 85.8 +/− 4.0 71.3 +/−6.8 E240N Y[155]F/K63N/I86S/R150Y/ 26-29 2 82.1 +/− 2.7 83.6 +/− 3.765.6 +/− 5.5 R170E/R233E/E240N N95S/R150Y/R170E/T175R/ 24-29 2 75.5 +/−4.5 76.6 +/− 4.3 82.2 +/− 5.8 R233E/E240N Y[155]F/N95S/R150Y/R170E/21-27 2 85.2 +/− 2.5 89.7 +/− 3.6 46.5 +/− 7.0 T175R/R233E/E240NK63N/K82N/N84S/R150Y/ 34-45 3 83.9 +/− 1.8 79.8 +/− 3.6 75.2 +/− 4.9R170E/T175R/R233E/E240N Y[155]F/K63N/K82N/N84S/ 24-26 2 84.6 +/− 3.4R150Y/R170E/T175R/R233E/ E240N Y[155]F/R170E/T175R/R233E 22-30 2 81.9+/− 3.6 79.2 +/− 6.2 55.0 +/− 8.0 R170E/T175R/R233E 23-28 2 60.6 +/− 6.486.5 +/− 4.3 35.6 +/− 8.3 Y[155]F/R170E/T175R/R233E/ 24-27 2 71.2 +/−4.5 77.8 +/− 5.3 54.6 +/− 8.2 E240S Y[155]F/N95S/R170E/T175R/ 25-29 258.2 +/− 7.9 65.5 +/− 8.3 48.2 +/− 10.0 R233E Y[155]F/I86S/R170E/T175R/23-30 2 84.1 +/− 5.1 90.9 +/− 2.7 76.6 +/− 6.4 R233ER150Y/R170E/T175R/R233E/ 27-43 3 80.2 +/− 2.5 87.1 +/− 3.2 76.9 +/− 4.0E240S Y[155]F/K82N/N84S/T175R/ 12-29 2 70.5 +/− 6.9 84.2 +/− 5.4 53.2+/− 12.3 39.5 +/− 11.1 R233E Y[155]F/K82N/N84S/R150Y/ 36-41 3 79.6 +/−3.2 90.5 +/− 2.4 73.8 +/− 4.6 R170E/T175R/R233E K82N/N84S/R150Y/R170E/22-28 2 84.3 +/− 3.1 91.8 +/− 1.4 60.1 +/− 6.7 T175R/R233EY[155]F/K82N/N84S/R170E/ 25-30 2 91.1 +/− 1.8 T175R/R233E/E240NK82N/N84S/R170E/T175R/ 25-28 2 82.7 +/− 4.5 R233E/E240NY[155]F/K82N/N84S/R150Y/ 20-29 2 83.3 +/− 3.9 R170EY[155]F/K82N/N84S/R150Y/ 24-28 2 43.6 +/− 6.5 T175RY[155]F/K82N/N84S/R170E/ 15-30 2 47.2 +/− 8.0 64.7 +/− 9.7 90.8 +/− 4.578.4 +/− 7.5 R233E Y[155]F/K82N/N84S/R170E/ 25-27 2 70.5 +/− 7.0 T175RY[155]F/K82N/N84S/R150Y/ 28-30 2 73.7 +/− 6.7 R170E/T175R/E240NK82N/N84S/R150Y/R170E/ 25-29 2 77.2 +/− 6.0 T175R/E240NY[155]F/K82N/N84S/R150Y/ 26-28 2 87.6 +/− 2.4 T175R/R233E/E240NK82N/N84S/R150Y/T175R/ 28-30 2 91.3 +/− 2.6 R233E/E240NY[155]F/K82N/N84S/R170E/ 25-30 2 74.6 +/− 6.4 E240NY[155]F/K82N/N84S/R150Y/ 27-30 2 85.2 +/− 4.4 T175R/R233EK82N/N84S/R150Y/T175R/ 25-30 2 51.9 +/− 8.2 E240NY[155]F/K82N/N84S/R170E/ 27-29 2 84.6 +/− 5.0 T175R/R233EK82N/N84S/R170E/T175R/ 27-29 2 73.0 +/− 6.6 R233E K82N/N84S/R170E/T175R/24-29 2 59.1 +/− 8.0 E240N Y[155]F/K82N/N84S/T175R/ 28-30 2 86.5 +/− 3.9R233E/E240N K82N/N84S/T175R/R233E/ 25-29 2 59.2 +/− 8.2 E240NY[155]F/T175R/R233E/E240N 24-28 2 78.7 +/− 4.9 Y[155]F/K82N/N84S/R150Y/28-30 2 82.3 +/− 5.4 R170E/T175R K82N/N84S/R150Y/R170E/ 37-43 3 79.3 +/−4.2 T175R R170E/T175R/E240N 37-41 3 66.6 +/− 5.9 R150Y/T175R/E240N 24-282 83.5 +/− 5.1 K63N/R150Y/R170E/T175R/ 23-29 2 84.5 +/− 3.1 R233E/E240NK63N/K82N/N84S/R150Y/ 22-28 2 81.9 +/− 4.1 R170E/T175R/R233E Inhibition(% of vehicle (0) +/− SEM) Mutation (chymotrypsin at each time point(hrs) numbering) 32 48 72 R150Y/R170E/R233E 71.8 +/− 7.0 40.2 +/− 7.8 7.8 +/− 5.2 R150Y/R170E/E240N 55.3 +/− 6.1 21.0 +/− 6.2 27.7 +/− 7.3Y[155]F/R150Y/R170E/E240N 56.8 +/− 9.0 15.6 +/− 8.2 R150Y/R233E/E240N15.8 +/− 4.3  4.8 +/− 5.4 −0.4 +/− 2.9 R150Y/R170E/R233E/E240N 63.6 +/−4.4 31.1 +/− 4.9  3.5 +/− 2.7 Y[155]F/R150Y/R170E/R233E/ 22.2 +/− 8.2−17.6 +/− 3.6  E240N D[104]N/K[106]S/Y[155]F/ 48.3 +/− 7.2 27.3 +/− 5.712.1 +/− 3.0 R150Y/R170E/R233E/E240N T175R 12.3 +/− 6.1 14.8 +/− 7.1 3.4 +/− 3.1 Y[155]F/K63N/R150Y/R170E/ 42.4 +/− 8.3 41.2 +/− 7.6  4.7+/− 5.6 R233E/E240N K82N/N84S/R150Y/R170E/ 49.9 +/− 6.8 31.4 +/− 5.9 5.0 +/− 4.2 R233E/E240N Y[155]F/K82N/N84S/R150Y/ 61.1 +/− 4.8 38.2 +/−5.2 17.1 +/− 4.0 R170E/R233E/E240N D[104]N/K[106]S/Y[155]F/K82N/ 48.7+/− 8.2 54.1 +/− 7.7 12.3 +/− 6.5 N84S/R150Y/R170E/R233E/ E240NK82N/N84S/R150Y/R170E/ 26.2 +/− 8.7 22.3 +/− 7.1 E240NY[155]F/K82N/N84S/R150Y/ 16.0 +/− 7.3 −2.2 +/− 4.3 R170E/E240NR150Y/R170E/R233E/E240S 48.3 +/− 6.5 12.3 +/− 5.3 −5.6 +/− 2.4K63N/K82N/N84S/R150Y/ 53.9 +/− 7.1 42.9 +/− 6.9 10.4 +/− 5.4R170E/R233E/E240N D[104]N/K[106]S/K63N/K82N/ 59.7 +/− 7.7 33.8 +/− 8.326.2 +/− 5.8 N84S/R150Y/R170E/R233E/ E240N Y[155]F/K63N/K82N/N84S/ 58.4+/− 6.3 27.0 +/− 7.7 14.1 +/− 7.8 R150Y/R170E/R233E/E240NR150Y/R170E/R233E/E240N/ 42.8 +/− 6.2  9.0 +/− 6.6 −3.8 +/− 3.4 T242VR150Y/R170E/R233E/E240N/ 35.6 +/− 6.3 29.3 +/− 6.0  3.7 +/− 3.1 T242AK82N/N84S/N95S/R150Y/R170E/ 16.0 +/− 4.7 0.08 +/− 3.8 −6.1 +/− 2.4R233E/E240N Y[155]F/K82N/N84S/N95S/ 44.3 +/− 6.2 27.0 +/− 6.4  8.1 +/−5.3 R150Y/R170E/R233E/E240N R150Y/R170E/T175R/R233E/ 69.4 +/− 6.0 23.2+/− 7.2  4.9 +/− 5.8 E240N Y[155]F/R150Y/R170E/T175R/ 66.7 +/− 6.6 56.8+/− 8.0  8.2 +/− 8.0 R233E/E240N D[104]N/K[106]S/R150Y/R170E/ 60.4 +/−7.9 54.8 +/− 6.7 12.8 +/− 6.3 T175R/R233E/E240N R150Y/R170E/T175R/N178Y/47.9 +/− 6.8 14.9 +/− 6.2 −12.1 +/− 2.9  R233E/E240NY[155]F/K82N/N84S/R150Y/ 61.1 +/− 7.4 46.0 +/− 6.9  0.4 +/− 4.9R170E/R233E K82N/N84S/R150Y/R170E/ 18.8 +/− 6.3  2.1 +/− 2.7 R233EY[155]F/K82N/N84S/R170E/ 58.4 +/− 5.8 51.1 +/− 6.6 26.9 +/− 5.4R233E/E240N K82N/N84S/R170E/R233E/ 16.7 +/− 6.3 −7.0 +/− 2.0 E240NR150Y/R170E/T175R/R233E 62.6 +/− 5.3 50.5 +/− 5.9  1.9 +/− 4.0Y[155]F/R150Y/R170E/T175R/ 67.2 +/− 7.3 52.2 +/− 8.2 41.1 +/− 8.3 R233ER150Y/R170E/T175R/E240N 53.1 +/− 6.8 46.4 +/− 6.7 34.9 +/− 6.0R150Y/T175R/R233E/E240N 39.4 +/− 8.1 24.5 +/− 7.6  6.8 +/− 4.8Y[155]F/R150Y/T175R/R233E/ 29.3 +/− 6.1  7.7 +/− 3.2 E240NR170E/T175R/R233E/E240N 47.2 +/− 8.6 33.0 +/− 8.4  9.2 +/− 5.3Y[155]F/R170E/T175R/R233E/ 61.3 +/− 5.8 35.6 +/− 8.3  5.9 +/− 5.0 E240NY[155]F/K82N/N84S/R150Y/ 64.2 +/− 5.4 51.4 +/− 5.7 33.1 +/− 7.3R170E/T175R/R233E/E240N K82N/N84S/R150Y/R170E/ 57.1 +/− 5.7 48.5 +/− 6.116.9 +/− 6.8 T175R/R233E/E240N K63N/I86S/R150Y/R170E/R233E/ 54.8 +/− 7.340.3 +/− 10.3  23.1 +/− 10.4 E240N Y[155]F/K63N/I86S/R150Y/ 57.2 +/− 7.938.4 +/− 8.9 16.5 +/− 7.7 R170E/R233E/E240N N95S/R150Y/R170E/T175R/ 84.7+/− 3.9 41.6 +/− 8.6 20.1 +/− 6.0 R233E/E240N Y[155]F/N95S/R150Y/R170E/63.3 +/− 8.0 41.6 +/− 8.8  9.1 +/− 6.5 T175R/R233E/E240NK63N/K82N/N84S/R150Y/ 80.9 +/− 3.0 73.0 +/− 4.4 43.8 +/− 6.6R170E/T175R/R233E/E240N Y[155]F/K63N/K82N/N84S/ 70.6 +/− 7.5 50.9 +/−8.6 R150Y/R170E/T175R/R233E/ E240N Y[155]F/R170E/T175R/R233E 44.4 +/−9.9 26.8 +/− 6.8 −6.5 +/− 2.7 R170E/T175R/R233E 35.8 +/− 8.5 18.9 +/−6.8 12.1 +/− 6.0 Y[155]F/R170E/T175R/R233E/ 58.3 +/− 8.1 21.9 +/− 7.1−11.0 +/− 3.4  E240S Y[155]F/N95S/R170E/T175R/ 29.3 +/− 9.3 21.0 +/− 6.7−14.8 +/− 5.3  R233E Y[155]F/I86S/R170E/T175R/ 62.4 +/− 6.7 55.2 +/− 7.923.7 +/− 6.5 R233E R150Y/R170E/T175R/R233E/ 67.9 +/− 5.6 48.3 +/− 5.521.0 +/− 5.0 E240S Y[155]F/K82N/N84S/T175R/ 18.0 +/− 7.3 17.0 +/− 5.4−7.4 +/− 3.1 R233E Y[155]F/K82N/N84S/R150Y/ 75.0 +/− 5.0 74.4 +/− 4.727.5 +/− 6.5 R170E/T175R/R233E K82N/N84S/R150Y/R170E/ 54.0 +/− 8.1 43.6+/− 8.8 35.7 +/− 8.7 T175R/R233E Y[155]F/K82N/N84S/R170E/ 22.7 +/− 6.612.8 +/− 6.2 T175R/R233E/E240N K82N/N84S/R170E/T175R/ 67.1 +/− 7.7 21.6+/− 8.0 R233E/E240N Y[155]F/K82N/N84S/R150Y/ 47.8 +/− 7.0 19.4 +/− 6.2R170E Y[155]F/K82N/N84S/R150Y/  4.9 +/− 4.6  7.2 +/− 1.9 T175RY[155]F/K82N/N84S/R170E/ 49.2 +/− 11.5 19.7 +/− 7.9 −5.8 +/− 4.2 R233EY[155]F/K82N/N84S/R170E/ 34.0 +/− 7.4 27.9 +/− 6.4 T175RY[155]F/K82N/N84S/R150Y/ 30.1 +/− 8.4 43.1 +/− 7.9 R170E/T175R/E240NK82N/N84S/R150Y/R170E/ 29.5 +/− 7.2 29.0 +/− 5.5 T175R/E240NY[155]F/K82N/N84S/R150Y/ 42.6 +/− 8.6 14.5 +/− 6.4 T175R/R233E/E240NK82N/N84S/R150Y/T175R/ 52.4 +/− 7.7  6.6 +/− 4.5 R233E/E240NY[155]F/K82N/N84S/R170E/ 30.1 +/− 7.1 12.4 +/− 6.3 E240NY[155]F/K82N/N84S/R150Y/ 31.1 +/− 7.8 −7.9 +/− 2.6 T175R/R233EK82N/N84S/R150Y/T175R/  9.4 +/− 4.9  3.2 +/− 4.5 E240NY[155]F/K82N/N84S/R170E/ 26.8 +/− 8.5 10.9 +/− 6.9 T175R/R233EK82N/N84S/R170E/T175R/ 27.3 +/− 7.7 23.4 +/− 5.6 R233EK82N/N84S/R170E/T175R/ 29.6 +/− 7.4 12.2 +/− 5.2 E240NY[155]F/K82N/N84S/T175R/ 34.6 +/− 8.1 −2.3 +/− 4.0 R233E/E240NK82N/N84S/T175R/R233E/  1.0 +/− 4.0 −7.3 +/− 2.8 E240NY[155]F/T175R/R233E/E240N −5.7 +/− 2.8 −4.2 +/− 3.7Y[155]F/K82N/N84S/R150Y/ 64.6 +/− 7.4 41.4 +/− 7.7 R170E/T175RK82N/N84S/R150Y/R170E/ 47.7 +/− 5.3 20.9 +/− 5.5 T175R R170E/T175R/E240N31.5 +/− 6 10.4 +/− 3.6 R150Y/T175R/E240N 36.7 +/− 8.9 20.0 +/− 6.7K63N/R150Y/R170E/T175R/ 66.3 +/− 7.8 41.2 +/− 8.5 R233E/E240NK63N/K82N/N84S/R150Y/ 62.2 +/− 8.2 28.6 +/− 8.0 R170E/T175R/R233E

Example 8 Determination of the Functional Cofactor Binding (K_(D-app))of FIXa for its Cofactor, Factor VIIIa

The functional cofactor binding (K_(D-app)) of the FIXa variants for thecofactor Factor VIIIa (FVIIIa) in the presence or saturating substrate,Factor X (FX), was assessed indirectly in a fluorogenic assay byassaying for the activity of FXa, generated upon activation by FIXa, onthe synthetic substrate Spectrafluor FXa. A range of FVIIIaconcentrations were used to calculate the apparent kinetic rate constant(K_(D-app)) where the cofactor (FVIIIa) was in excess by at least a1000-fold over the concentration of the activating protease (FIXa). Theexperiment was designed to be a variation of the assay described inExample 4 (Determination of the Catalytic Activity of FIXa for itsSubstrate, Factor X) where the cofactor (FVIIIa) at variousconcentrations is preincubated with FIXa in the presence of phospholipidvesicles forming the tenase (Xase) complex prior to assessing thecatalytic activity with saturating levels of the substrate, FX. Briefly,activated and active site titrated FIXa was incubated in acalcium-containing buffer with phospholipid vesicles while separatelyrecombinant FVIII is activated (to FVIIIa) with alpha-thrombin. Theactivity of alpha-thrombin was then quenched by the addition of a highlyspecific thrombin inhibitor, hirudin, prior to initiating the assay.FIXa variants were then mixed with various concentrations of FVIIIa toform the Xase complex and subsequently mixed with saturatingconcentrations of FX and the fluorescent substrate, Spectrafluor FXa(CH₃SO₂-D-CHA-Gly-Arg-AMC) to initiate the assay. The release of thefree fluorophore, AMC (7-amino-4-methylcoumarin) following catalysis ofSpectrafluor FXa by FXa was then assessed continuously over a timeperiod, and the kinetic rate constants of the FIXa variants determined.

A. Assay Protocol

For assays evaluating the kinetic rate of FX activation by FIXa in thepresence of various FVIIIa concentrations and phospholipids, recombinantFVIII (Kogenate FS®; Bayer healthcare) was first resuspended in 1 mL ofthe provided diluent. The molar concentration of FVIII was thendetermined by absorbance at 280 nm using an extinction coefficient of1.567 mg⁻¹ mL cm⁻¹ and a molecular weight of 163.6 kDa. The FIX variantswere expressed, purified, activated and active site titrated asdescribed in Examples 1-3, above. FIXa variants were then seriallydiluted to a concentration of 8 pM (4×) in a 1 mL volume of 1× Buffer A(20 mM Hepes/150 mM NaCl/5 mM CaCl₂/0.1% BSA/0.1% PEG-8000, pH 7.4). Inpreparation for activation of FVIII to FVIIIa in the presencephospholipids, alpha-thrombin (Heamatologic Technologies, Inc.) andhirudin (American Diagnostica) were each diluted from the manufacturer'sstock concentrations 1:100 in 1× Buffer A. Reconstituted FVIII wasfurther diluted to a concentration of 1600 nM (4× of the top dose) in a1.6 mL volume of 1× Buffer A containing 400 μM freshly resuspendedphospholipids (75% phosphatidylcholine (PC)/25% phospatidylserine (PS);PS/PC vesicles ˜120 nm in diameter; Avanti Polar Lipids). FVIII wasactivated to FVIIIa by mixing the above FVIII/PC/PS solution with afinal concentration of 15 nM alpha-thrombin solutions followed by 15minutes of incubation at 25° C. Activation reactions were subsequentlyquenched by the addition of hirudin to a final concentration of 150 nMfor 5 min at 25° C. prior to initiating a dilution series of 1.5-fold ina 12-channel deep-well polypropylene plate with a final volume of 0.5 mLof the activated FVIIIa into 1× Buffer A containing 400 μM PC/PSvesicles. The final concentrations of FVIIIa (4×) were 1600 nM, 1066.7nM, 711.1 nM, 474.1 nM, 316.1 nM, 210.7 nM, 140.5 nM, 93.6 nM, 62.43 nM,41.6 nM. 27.8 nM and 0 nM for a 12-point assay or for an alternatibe8-point assay with a 2-fold dilution series; 1600 nM, 600 nM, 400 nM,200 nM, 100 nM, 50 nM, 25 nM and 0 nM. The dilution series of FVIIIa wassubsequently mixed 1:1 with the 4×FIXa dilutions (12.5 μL each) in a96-well half-area black assay plate according to a predefined plate map(4 FIXa variants/plate) and preincubated 15 min at 25° C. to form Xasecomplexes with varied concentrations of FVIIIa. Final 2× solutions (25μL) were as follows: 4 pM FIXa variant, 1600-0 nM FVIIIa, 200 μM PC/PSvesicles, 7.5 nM alpha-thrombin (inhibited) and 75 nM hirudin.

A solution of 1000 nM (2×) active site titrated and DFP/EGR-cmk treatedFX (see Example 2, above) was prepared in 20 mL of 1× Buffer Acontaining 1.0 mM Spectrafluor Xa substrate providing a sufficientvolume for 4 assays. This represented a 2× saturating concentration ofFX that would be at least 5-20-fold above the K_(M) values reported inExample 4, Table 16. Assay reactions were typically initiated using aBioMek FX liquid handling system programmed to dispense 25 μL of theFX/Spectrafluor Xa dilutions into 4 assay plates containing 25 μL ofeach FIXa variant and FVIIIa dilution (Xase complexes). The finalconcentrations of the reagents in the assay were as follows: 2 pM FIXa,400-0 nM FVIIIa, 100 μM PC/PS vesicles, 0.5 mM Spectrafluor Xa, 3.8 nMalpha thrombin (inhibited), 38 nM hirudin and FX at 500 nM. Reactionswere monitored in a SpectraMax fluorescence plate reader for 30 min at37° C. A standard curve of free AMC served as the conversion factor forRFU to μM in the subsequent data analysis calculations using a doserange that covered 0 μM to 100 μM AMC.

B. Data Analysis

To determine functional affinity of FIXa variants for FVIIIa based ontheir catalytic activity, raw data collected with the SoftMax Proapplication (Molecular Devices) were exported as .TXT files. Furthernon-linear data analyses were performed directly within the ActivityBasesoftware package using the XE Runner data analysis module (IDBSSoftware). Data analyses were essentially as described in Example 4Bwith minor modifications. The Abase template was set up to automaticallyfit the parabolic reaction velocities (μM/sec²) of the tested FIXavariants at each FVIIIa concentration to the function of a standardrectangular hyperbola (i.e. Michaelis Menten equation) given by equation(1) to yield the fit values for V_(max) and K_(D-app).

$\begin{matrix}{{{Reaction}\mspace{14mu} {{Velocity}\left( {{µM}\text{/}\sec^{2}} \right)}} = \frac{V_{\max}\left\lbrack S_{0} \right\rbrack}{K_{D - {app}} + \left\lbrack S_{0} \right\rbrack}} & {{Equation}\mspace{14mu} (1)}\end{matrix}$

Table 31 sets forth the functional affinity (K_(D-app)) for each of theFIXa variants assayed. Also assayed were recombinant wild-type FIXa(termed Catalyst Biosciences WT; generated as described above in Example1), plasma purified FIXa (Haematologic Technologies, Inc.), and BeneFIX®(Coagulation Factor IX (Recombinant); Wyeth). Table XX presents theresults expressed as the kinetic constant for affinity, K_(D-app) (nM),and also as ratio of the functional affinity of the wild-type FIXacompared to that of the FIXa variant, wherein the functional affinity ofeach FIXa variant is defined by the K_(D-app) (nM) value for activationof the substrate, FX. Where the activity of the FIXa variant wascompared to wild-type FIXa, it was compared to a recombinant wild-typeFIXa polypeptide that was expressed and purified using the sameconditions as used for the variant FIXa polypeptides to ensure that anydifferences in activity were the result of the mutation(s), and not theresult of differences in, for example, post-translational modificationsassociated with different expression systems. Thus, the wild-type FIXapolypeptide used for comparison was the recombinant wild-type FIXagenerated from cloning the FIX gene set forth in SEQ ID NO:1 andexpressed from CHOX cells as a polypeptide with an amino acid sequenceset forth in SEQ ID NO:3, as described in Example 1 (i.e. CatalystBiosciences WT FIX polypeptide). The standard deviation (S.D.),coefficient of variation (as a percentage; % CV) and the number ofassays performed (n) also are provided.

While some variants showed similar to wild-type affinities or nominalincreases in K_(D-app) (e.g. FIXa-R318Y/R338E andFIXa-R318Y/R338E/R403E/E410N) several variants showed marked increasesin functional affinity with greater than 6-10 fold increases inK_(D-app) Variants with combinations of the R338E, T343R and E410Nmutations showed the greatest improvements in functional affinity. Forinstance, FIXa-R338E/T343R, FIXa-R318Y/R338E/T343R/E410N,FIXa-R318Y/R338E/E410N,FIXa-Y155F/K247N/N249S/R318Y/R338E/T343R/R403E/E410N, FIXa-R338E/E410Nand FIXa-K228N/247N/N249S/R318Y/R338E/T343R/E410N are among this group.

TABLE 31 Functional Cofactor Affinity of FIXa variants (K_(D-app))Mutation (Mature FIX Mutation (Chymotrypsin K_(D-app) ±S.D. K_(D-WT)/Numbering) Numbering) (nM) (nM) % CV K_(D-mut) n BeneFIX Benefix ®Coagulation BeneFIX Benefix ® Coagulation 90.2 13.5 15% 1.1 4 FIX(T148A) FIX (T[148]A) Plasma Purified FIXa Plasma Purified FIXa 101.65.8 6% 0.9 3 Catalyst Biosciences WT Catalyst Biosciences WT 95.5 4.6 5%1.0 2 T148A T[148]A 79.7 27.1 34% 1.2 2 D104N/K106S/I251SD[104]N/K[106]S/I86S 305.5 119.5 39% 0.3 2 A262S A95bS 94.1 18.3 19% 1.02 E410N E240N 74.2 0.6 1% 1.3 2 E239N E74N 77.3 40.6 53% 1.2 2T241N/H243S T76N/H78S 75.5 26.2 35% 1.3 2 S319N/L321S S151N/L153S 52.40.7 1% 1.8 2 R318E R150E 67.0 5.2 8% 1.4 2 R318Y R150Y 192.0 55.2 29%0.5 2 R312Q R143Q 45.2 5.6 12% 2.1 2 R312A R143A 52.9 5.9 11% 1.8 2R312Y R143Y 85.2 36.5 43% 1.1 2 R312L R143L 68.9 15.6 23% 1.4 2 V202YV38Y 61.5 3.5 6% 1.6 2 D203Y D39Y 77.4 11.8 15% 1.2 2 A204M A40M 60.69.0 15% 1.6 2 K400A/R403A K230A/R233A 129.5 13.4 10% 0.7 2 K400E/R403EK230E/R233E 298.0 58.0 19% 0.3 2 R403E R233E 654.0 131.6 20% 0.1 3 K400AK230A 98.9 7.2 7% 1.0 2 K293A K126A 86.6 4.0 5% 1.1 2 R338E R170E 43.07.2 17% 2.2 2 R338E/R403E R170E/R233E 183.0 42.4 23% 0.5 2 R338E/E410NR170E/E240N 4.1 1.4 33% 23.5 3 R338E/R403E/E410N R170E/R233E/E240N 54.93.0 6% 1.7 2 R318Y/R338E/R403E R150Y/R170E/R233E 340.0 244.7 72% 0.3 2R403E/E410N R233E/E240N 910.5 197.3 22% 0.1 2 R318Y/R338E/E410NR150Y/R170E/E240N 7.7 4.6 60% 12.4 17 D104N/K106S/R318Y/R338E/D[104]N/K[106]S/R150Y/R170E/ 12.4 n.d. n.d. 7.7 1 E410N E240NR318Y/R338E/R403E/E410N R150Y/R170E/R233E/E240N 47.0 12.4 26% 2.0 12D104N/K106S/Y155F/R318Y/ D[104]N/K[106]S/Y[155]F/ 61.6 n.d. n.d. 1.6 1R338E/R403E/E410N R150Y/R170E/R233E/E240N K316N K148N 66.4 8.3 13% 1.4 2H257E H92E 81.3 2.5 3% 1.2 2 E410S E240S 99.6 2.0 2% 1.0 2 N346D N178D126.5 3.5 3% 0.8 2 N346Y N178Y 65.7 n.d. n.d. 1.5 1 Y345A Y177A 29.6 2.38% 3.2 2 T343R T175R 58.4 16.2 28% 1.6 3 T343R/Y345T T175R/Y177T 68.1n.d. n.d. 1.4 1 R318Y/R338E R150Y/R170E 28.9 n.d. n.d. 3.3 1Y259F/K265T/Y345T Y94F/K98T/Y177T 115.2 n.d. n.d. 0.8 1 K228N/I251SK63N/I86S 89.7 1.3 1% 1.1 2 Y155F/K228N/R318Y/R338E/Y[155]F/K63N/R150Y/R170E/ 31.2 4.8 15% 3.1 2 R403E/E410N R233E/E240NI251S/R318Y/R338E/R403E/ I86S/R150Y/R170E/R233E/ 62.7 0.6 1% 1.5 2 E410NE240N D104N/K106S/I251S/R318Y/ D[104]N/K[106]S/I86S/R150Y/ 54.7 19.9 36%1.7 5 R338E/R403E/E410N R170E/R233E/E240N I251S/R318Y/R338E/E410NI86S/R150Y/R170E/E240N 5.7 1.1 20% 16.7 3 D104N/K106S/I251S/R318Y/D[104]N/K[106]S/I86S/R150Y/ 12.4 1.1 9% 7.7 2 R338E/E410N R170E/E240NK247N/N249S/R318Y/R338E/ K82N/N84S/R150Y/R170E/ 68.6 17.3 25% 1.4 3R403E/E410N R233E/E240N Y155F/K247N/N249S/R318Y/Y[155]F/K82N/N84S/R150Y/ 45.8 4.6 10% 2.1 7 R338E/R403E/E410NR170E/R233E/E240N A103N/N105S/K247N/N249S/ A[103]N/N[105]S/K82N/N84S/93.1 8.4 9% 1.0 2 R318Y/R338E/R403E/E410N R150Y/R170E/R233E/E240ND104N/K106S/K247N/N249S/ D[104]N/K[106]S/K82N/N84S/ 87.4 10.3 12% 1.1 2R318Y/R338E/R403E/E410N R150Y/R170E/R233E/E240N Y155F/K247N/N249S/R318Y/Y[155]F/K82N/N84S/R150Y/ 7.4 n.d. n.d. 12.8 1 R338E/E410N R170E/E240NR318Y/R338E/R403E/E410S R150Y/R170E/R233E/E240S 53.1 10.4 20% 1.8 3R318Y/R338E/E410S R150Y/R170E/E240S 6.8 0.2 3% 14.1 3 K228N/K247N/N249SK63N/K82N/N84S 113.0 0.0 0% 0.8 2 K228N/K247N/N249S/R318Y/K63N/K82N/N84S/R150Y/R170E/ 100.5 n.d. n.d. 0.9 1 R338E/R403E/E410NR233E/E240N R318Y/R338E/R403E/E410N/ R150Y/R170E/R233E/E240N/ 55.0 n.d.n.d. 1.7 1 T412V T242V R318Y/R338E/E410N/T412V R150Y/R170E/E240N/T242V8.9 n.d. n.d. 10.7 1 R318Y/R338E/N346D/R403E/ R150Y/R170E/N178D/R233E/109.7 44.3 40% 0.9 2 E410N E240N K247N/N249S/N260S K82N/N84S/N95S 147.060.8 41% 0.6 2 Y155F/K247N/N249S/N260S Y[155]F/K82N/N84S/N95S 167.0 97.758% 0.6 2 D104N/K106S/K247N/N249S/ D[104]N/K[106]S/K82N/N84S/ 330.0319.6 97% 0.3 2 N260S N95S D104N/K106S/Y155F/K247N/D[104]N/K[106]S/Y[155]F/K82N/ 142.0 73.5 52% 0.7 2 N249S/N260S N84S/N95SK247N/N249S/N260S/R318Y/ K82N/N84S/N95S/R150Y/R170E/ 65.0 10.8 17% 1.5 2R338E/R403E/E410N R233E/E240N R318Y/R338E/T343R/R403E/R150Y/R170E/T175R/R233E/ 14.5 4.0 28% 6.6 7 E410N E240N R338E/T343RR170E/T175R 3.4 0.6 18% 28.0 2 T343R/N346Y T175R/N178Y 38.6 n.d. n.d.2.5 1 R318Y/R338E/N346Y/R403E/ R150Y/R170E/N178Y/R233E/ 39.6 n.d. n.d.2.4 1 E410N E240N R318Y/R338E/T343R/N346Y/ R150Y/R170E/T175R/N178Y/ 15.60.1 1% 6.1 2 R403E/E410N R233E/E240N T343R/N346D T175R/N178D 78.4 n.d.n.d. 1.2 1 R318Y/R338E/T343R/N346D/ R150Y/R170E/T175R/N178D/ 76.2 n.d.n.d. 1.3 1 R403E/E410N R233E/E240N R318Y/R338E/T343R/E410NR150Y/R170E/T175R/E240N 6.1 n.d. n.d. 15.7 1 Y155F/R318Y/R338E/T343R/Y[155]F/R150Y/R170E/T175R/ 7.4 n.d. n.d. 12.8 1 E410N E240NR318Y/T343R/R403E/E410N R150Y/T175R/R233E/E240N 84.1 17.8 21% 1.1 2R338E/T343R/R403E/E410N R170E/T175R/R233E/E240N 29.4 n.d. n.d. 3.2 1Y155F/R338E/T343R/R403E/ Y[155]F/R170E/T175R/R233E/ 28.5 n.d. n.d. 3.3 1E410N E240N Y155F/K247N/N249S/R318Y/ Y[155]F/K82N/N84S/R150Y/ 15.3 1.39% 6.3 3 R338E/T343R/R403E/E410N R170E/T175R/R233E/E240NK228N/K247N/N249S/R318Y/ K63N/K82N/N84S/R150Y/R170E/ 29.1 0.3 1% 3.3 2R338E/T343R/R403E/E410N T175R/R233E/E240N Y155F/K228N/K247N/N249S/R318Y/Y[155]F/K63N/K82N/N84S/R150Y/ 37.0 5.7 16% 2.6 2 R338E/T343R/R403E/E410NR170E/T175R/R233E/E240N Y155F/R338E/T343R/R403EY[155]F/R170E/T175R/R233E 72.1 n.d. n.d. 1.3 1 R338E/T343R/R403ER170E/T175R/R233E 55.0 n.d. n.d. 1.7 1 R318Y/R338E/T343R/R403E/R150Y/R170E/T175R/R233E/ 23.2 n.d. n.d. 4.1 1 E410S E240SY155F/K247N/N249S/R338E/ Y[155]F/K82N/N84S/R170E/ 15.4 n.d. n.d. 6.2 1T343R T175R Y155F/K247N/N249S/R318Y/ Y[155]F/K82N/N84S/R150Y/ 13.9 n.d.n.d. 6.9 1 R338E/T343R/E410N R170E/T175R/E240N Y155F/K247N/N249S/R338E/Y[155]F/K82N/N84S/R170E/ 24.9 n.d. n.d. 3.8 1 E410N E240NK247N/N249S/R338E/T343R/ K82N/N84S/R170E/T175R/ 14.0 n.d. n.d. 6.8 1E410N E240N Y155F/R318Y/R338E/T343R Y[155]F/R150Y/R170E/T175R 8.4 n.d.n.d. 11.3 1 R318Y/R338E/T343R R150Y/R170E/T175R 9.8 n.d. n.d. 9.7 1Y155F/K247N/N249S/R318Y/ Y[155]F/K82N/N84S/R150Y/ 14.0 n.d. n.d. 6.8 1R338E/T343R R170E/T175R K247N/N249S/R318Y/R338E/ K82N/N84S/R150Y/R170E/14.7 n.d. n.d. 6.5 1 T343R T175R Y155F/R338E/T343R/E410NY[155]F/R170E/T175R/E240N 8.5 n.d. n.d. 11.2 1 R338E/T343R/E410NR170E/T175R/E240N 7.5 n.d. n.d. 12.8 1 Y155F/R318Y/T343R/E410NY[155]F/R150Y/T175R/E240N 38.0 n.d. n.d. 2.5 1 K228N/R150Y/R338E/T343R/K63N/R150Y/R170E/T175R/ 17.5 n.d. n.d. 5.4 1 R403E/E410N R233E/E240NK228N/247N/N249S/R318Y/ K63N/K82N/N84S/R150Y/R170E/ 7.8 n.d. n.d. 12.2 1R338E/T343R/E410N T175R/E240N

Example 9 Determination of the Clotting Activities of FIX Variants inHemophilia B Plasma

Clotting activities for FIX variants were determine by an activatedpartial thromboplastin time (aPTT) assay in human hemophilia B plasmafrom a single donor with <1% clotting activity (George King Bio-Medical,Inc., Overland Park, Kans.) per the manufacturer's instructions.Briefly, the aPTT assay involves the recalcification of plasma in thepresence of a blend of purified phospholipids (platelet substitute) andactivators (kaolin and sulphatide). The aPTT assay was performed usingthe Dapttin®TC aPTT reagent (Technoclone GmbH, Vienna, Austria)essentially as described in the manufacturers' product insert with FIXvariants spiked into the hemophilia B plasma at final concentrations of100 nM, 10 nM or 1 nM FIX variant. Briefly, FIX variants were diluted to1 μM in 1× Buffer A (20 mM Hepes/150 mM NaCl/0.5% BSA, pH 7.4) based onthe active site titrated zymogen concentration (Example 2). FIX variantswere subsequently serially diluted to 100 nM, 10 nM and 1 nM directlyinto citrated human hemophilia B plasma (George King Bio-Medical). A 100μL volume of each FIX dilution in plasma was mixed with 100 μL of theDapttin®TC aPTT reagent and incubated at 37° C. for 180 seconds.Coagulation was initiated by the addition of 100 μL of 25 mM calcium(Diagnostica Stago, Asnieres, France). Coagulation time in seconds wasmeasured using a STArt4 instrument (Diagnostica Stago, Asnieres,France). Each experiment represents the average of two independentclotting time measurements, which typically showed <5% CV.

Table 32 sets forth the clotting activities for each of the FIX variantsassayed. Also assayed were recombinant wild-type FIX (termed CatalystBiosciences WT; generated as described above in Example 1), and BeneFIX®(Coagulation Factor IX (Recombinant); Wyeth). Table XX presents theresults expressed as the time to clot at each of the three tested FIXconcentrations; 100 nM, 10 nM and 1 nM, wherein each FIX concentrationrepresents ˜100%, ˜10% and ˜1% of the normal concentration of FIX inpooled normal plasma (PNP). Under identical assay conditions, 100% PNPshows a clotting time of 31.3±2.0 seconds, whereas clotting times for10% and 1% dilutions of PNP in hemophilia B plasma are 42.7±1.7 and55.0±4.7 seconds, respectively (n=4). The time to clot for thehemophilia B plasma used in these analyses was evaluated 83.2±9.2seconds (n=5). A number of tested variants demonstrated clotting timessimilar to or slightly prolonged compared to the wild-type FIXa, wherewild-type FIXa polypeptide used for comparison was the recombinantwild-type FIXa expressed from CHOX cells as a polypeptide with an aminoacid sequence set forth in SEQ ID NO:3, as described in Example 1 (i.e.Catalyst Biosciences WT FIX polypeptide). On the other hand, severalvariants showed significantly shortened clotting times. Among this groupof variants are FIXa-R318Y/R338E/T343R, FIXa-R318Y/R338E/E410N,FIXa-R338E/T343R/E410N, FIXa-R318Y/R338E/T343R/E410N,FIXa-K247N/N249S/R338E/T343R/E410N andFIXa-K228N/247N/N249S/R318Y/R338E/T343R/E410N.

TABLE 32 Clotting Activity (aPTT) of FIX Variants in Hemophilia B PlasmaMutation aPTT aPTT aPTT Mutation (Mature (Chymotrypsin (100 nM) (10 nM)(1.0 nM) FIX Numbering) Numbering) (s) ±S.D. (s) ±S.D. (s) ±S.D. nBeneFIX Benefix ® BeneFIX Benefix ® 35.2 n.d. 47.4 n.d. 63.5 n.d. 1Coagulation FIX Coagulation FIX (T148A) (T[148]A) Catalyst BiosciencesCatalyst Biosciences WT 35.5 n.d. 46.9 n.d. 60.6 n.d. 1 WT T148A T[148]A33.2 n.d. 43.1 n.d. 59.2 n.d. 1 R338E/R403E R170E/R233E 34.3 n.d. 46.2n.d. 58.8 n.d. 1 R338E/R403E/E410N R170E/R233E/E240N 35.6 n.d. 46.6 n.d.57.1 n.d. 1 Y155F/R338E/R403E/ Y[155]F/R170E/R233E/ 31.1 n.d. 41.2 n.d.52.6 n.d. 1 E410N E240N R318Y/R338E/R403E R150Y/R170E/R233E 41.7 n.d.52.7 n.d. 68.4 n.d. 1 Y155F/R318Y/R338E/ Y[155]F/R150Y/R170E/ 38.6 n.d.48.6 n.d. 64.1 n.d. 1 R403E R233E R318Y/R338E/E410N R150Y/R170E/E240N21.2 n.d. 24.8 n.d. 34.3 n.d. 1 D104N/K106S/R318Y/D[104]N/K[106]S/R150Y/ 24.5 n.d. 30.8 n.d. 40.0 n.d. 1 R338E/E410NR170E/E240N R318Y/R403E/E410N R150Y/R233E/E240N 46.1 n.d. 61.7 n.d. 78.3n.d. 1 Y155F/R318Y/R403E/ Y[155]F/R150Y/R233E/ 42.3 n.d. 57.1 n.d. 74.5n.d. 1 E410N E240N R318Y/R338E/R403E/ R150Y/R170E/R233E/E240N 25.4 1.233.0 2.1 43.0 1.1 3 E410N T343R T175R 41.3 2.1 53.3 2.9 67.2 6.2 2T343R/Y345T T175R/Y177T 46.8 2.8 56.3 9.6 75.5 1.8 2 R318Y/R338ER150Y/R170E 26.7 n.d. 31.5 n.d. 45.3 n.d. 1 Y155F/K228N/R318Y/Y[155]F/K63N/R150Y/ 35.6 n.d. 45.1 n.d. 60.1 n.d. 1 R338E/R403E/E410NR170E/R233E/E240N D104N/K106S/I251S/R318Y/ D[104]N/K[106]S/I86S/ 36.0n.d. 46.8 n.d. 61.8 n.d. 1 R338E/R403E/E410N R150Y/R170E/R233E/ E240NI251S/R318Y/R338E/E410N I86S/R150Y/R170E/ 28.0 n.d. 30.1 n.d. 40.7 n.d.1 E240N D104N/K106S/I251S/R318Y/ D[104]N/K[106]S/I86S/ 25.0 n.d. 31.0n.d. 43.1 n.d. 1 R338E/E410N R150Y/R170E/E240N K247N/N249S/R318Y/K82N/N84S/R150Y/ 33.7 n.d. 43.8 n.d. 58.4 n.d. 1 R338E/R403E/E410NR170E/R233E/E240N Y155F/K247N/N249S/ Y[155]F/K82N/N84S/ 34.1 n.d. 46.2n.d. 62.4 n.d. 1 R318Y/R338E/R403E/ R150Y/R170E/R233E/ E410N E240NA103N/N105S/K247N/ A[103]N/N[105]S/K82N/ 36.1 n.d. 48.1 n.d. 62.6 n.d. 1N249S/R318Y/R338E/ N84S/R150Y/R170E/ R403E/E410N R233E/E240ND104N/K106S/Y155F/ D[104]N/K[106]S/Y[155]F/ 34.8 n.d. 45.6 n.d. 59.3n.d. 1 K247N/N249S/R318Y/ K82N/N84S/R150Y/ R338E/R403E/E410NR170E/R233E/E240N K247N/N249S/R318Y/ K82N/N84S/R150Y/ 26.1 n.d. 34.3n.d. 44.7 n.d. 1 R338E/E410N R170E/E240N Y155F/K247N/N249S/Y[155]F/K82N/N84S/ 24.0 n.d. 29.2 n.d. 41.1 n.d. 1 R318Y/R338E/E410NR150Y/R170E/E240N R318Y/R338E/R403E/ R150Y/R170E/R233E/ 26.9 n.d. 34.7n.d. 47.0 n.d. 1 E410S E240S K228N/K247N/N249S K63N/K82N/N84S 44.4 n.d.57.2 n.d. 70.2 n.d. 1 D104N/K106S/Y155F/ D[104]N/K[106]S/Y[155]F/ 46.9n.d. 60.0 n.d. 73.6 n.d. 1 K228N/K247N/N249S K63N/K82N/N84SK228N/K247N/N249S/ K63N/K82N/N84S/ 35.3 5.1 46.1 8.0 60.6 8.9 2R318Y/R338E/R403E/ R150Y/R170E/R233E/ E410N E240N D104N/K106S/K228N/D[104]N/K[106]S/K63N/ 38.4 n.d. 50.1 n.d. 67.1 n.d. 1 K247N/N249S/R318Y/K82N/N84S/R150Y/ R338E/R403E/E410N R170E/R233E/E240N Y155F/K228N/K247N/Y[155]F/K63N/K82N/ 34.9 n.d. 44.7 n.d. 59.1 n.d. 1 N249S/R318Y/R338E/N84S/R150Y/R170E/ R403E/E410N R233E/E240N R318Y/R338E/R403E/R150Y/R170E/R233E/ 28.7 n.d. 37.6 n.d. 47.6 n.d. 1 E410N/T412VE240N/T242V R318Y/R338E/R403E/ R150Y/R170E/R233E/ 30.5 n.d. 40.6 n.d.52.8 n.d. 1 E410N/T412A E240N/T242A R318Y/R338E/E410N/R150Y/R170E/E240N/ 25.5 n.d. 30.7 n.d. 40.3 n.d. 1 T412V T242VR318Y/R338E/N346D/ R150Y/R170E/N178D/ 42.5 n.d. 54.2 n.d. 68.9 n.d. 1R403E/E410N R233E/E240N Y155F/R318Y/R338E/ Y[155]F/R150Y/R170E/ 37.8n.d. 48.9 n.d. 65.2 n.d. 1 N346D/R403E/E410N N178D/R233E/E240NK247N/N249S/N260S/ K82N/N84S/N95S/ 44.7 n.d. 56.9 n.d. 75.7 n.d. 1R318Y/R338E/R403E/ R150Y/R170E/R233E/ E410N E240N Y155F/K247N/N249S/Y[155]F/K82N/N84S/N95S/ 49.3 n.d. 59.6 n.d. 75.5 n.d. 1N260S/R318Y/R338E/ R150Y/R170E/R233E/ R403E/E410N E240NR318Y/R338E/T343R/ R150Y/R170E/T175R/R233E/ 23.7 2.7 29.7 3.3 39.7 6.5 4R403E/E410N E240N Y155F/R318Y/R338E/ Y[155]F/R150Y/R170E/ 26.2 3.6 32.03.9 42.4 1.8 2 T343R/R403E/E410N T175R/R233E/E240N D104N/K106S/R318Y/D[104]N/K[106]S/R150Y/ 27.3 n.d. 34.9 n.d. 48.0 n.d. 1R338E/T343R/R403E/ R170E/T175R/R233E/ E410N E240N R338E/T343RR170E/T175R 27.9 n.d. 33.8 n.d. 45.1 n.d. 1 T343R/N346Y T175R/N178Y 40.83.8 54.9 0.8 74.9 2.2 2 R318Y/R338E/N346Y/ R150Y/R170E/N178Y/ 28.8 n.d.41.0 n.d. 54.4 n.d. 1 R403E/E410N R233E/E240N R318Y/R338E/T343R/R150Y/R170E/T175R/N178Y/ 24.5 n.d. 32.5 n.d. 41.7 n.d. 1N346Y/R403E/E410N R233E/E240N T343R/N346D T175R/N178D 39.9 1.4 51.3 4.865.0 4.1 2 R318Y/R338E/T343R/ R150Y/R170E/T175R/ 34.8 n.d. 45.1 n.d.57.9 n.d. 1 N346D/R403E/E410N N178D/R233E/E240N R318Y/R338E/Y345A/R150Y/R170E/Y177A/ 41.2 n.d. 47.9 n.d. 61.9 n.d. 1 R403E/E410NR233E/E240N Y155F/K247N/N249S/ Y[155]F/K82N/N84S/ 40.2 n.d. 51.6 n.d.62.2 n.d. 1 R318Y/R338E/R403E R150Y/R170E/R233E K247N/N249S/R318Y/K82N/N84S/R150Y/ 42.0 n.d. 55.6 n.d. 70.3 n.d. 1 R338E/R403E R170E/R233EK247N/N249S/R318Y/ K82N/N84S/R150Y/ 44.6 3.0 57.2 4.2 71.5 6.1 3R403E/E410N R233E/E240N Y155F/K247N/N249S/ Y[155]F/K82N/N84S/ 31.0 n.d.42.1 n.d. 55.6 n.d. 1 R338E/R403E/E410N R170E/R233E/E240NK247N/N249S/R338E/ K82N/N84S/R170E/ 32.7 n.d. 42.2 n.d. 56.2 n.d. 1R403E/E410N R233E/E240N R318Y/R338E/T343R/ R150Y/R170E/T175R/ 30.1 n.d.37.9 n.d. 51.4 n.d. 1 R403E R233E Y155F/R318Y/R338E/Y[155]F/R150Y/R170E/ 32.0 n.d. 41.5 n.d. 53.7 n.d. 1 T343R/R403ET175R/R233E R318Y/R338E/T343R/ R150Y/R170E/T175R/E240N 24.7 2.9 27.2 2.936.5 3.8 5 E410N Y155F/R318Y/R338E/ Y[155]F/R150Y/R170E/ 25.9 2.1 28.83.5 38.5 4.2 2 T343R/E410N T175R/E240N R318Y/T343R/R403E/R150Y/T175R/R233E/ 31.7 n.d. 43.3 n.d. 60.7 n.d. 1 E410N E240NY155F/R318Y/T343R/ Y[155]F/R150Y/T175R/ 40.3 n.d. 52.0 n.d. 68.7 n.d. 1R403E/E410N R233E/E240N R338E/T343R/R403E/ R170E/T175R/R233E/ 25.5 n.d.30.4 n.d. 41.9 n.d. 1 E410N E240N Y155F/R338E/T343R/Y[155]F/R170E/T175R/ 27.5 n.d. 33.3 n.d. 42.3 n.d. 1 R403E/E410NR233E/E240N Y155F/K247N/N249S/ Y[155]F/K82N/N84S/R150Y/ 24.2 0.9 29.71.4 40.5 2.4 5 R318Y/R338E/T343R/ R170E/T175R/R233E/ R403E/E410N E240NK247N/N249S/R318Y/ K82N/N84S/R150Y/ 28.7 n.d. 36.2 n.d. 50.2 n.d. 1R338E/T343R/R403E/ R170E/T175R/R233E/ E410N E240N K228N/I251S/R318Y/K63N/I86S/R150Y/ 34.5 n.d. 44.9 n.d. 58.2 n.d. 1 R338E/R403E/E410NR170E/R233E/E240N Y155F/K228N/I251S/ Y[155]F/K63N/I86S/ 34.5 n.d. 46.5n.d. 60.3 n.d. 1 R318Y/R338E/R403E/ R150Y/R170E/R233E/ E410N E240NN260S/R318Y/R338E/ N95S/R150Y/R170E/T175R/ 31.4 n.d. 41.1 n.d. 55.4 n.d.1 T343R/R403E/E410N R233E/E240N Y155F/N260S/R318Y/ Y[155]F/N95S/R150Y/35.3 0.6 45.3 2.5 59.1 3.2 2 R338E/T343R/R403E/ R170E/T175R/R233E/ E410NE240N K228N/K247N/N249S/ K63N/K82N/N84S/R150Y/ 28.0 2.0 35.5 3.9 47.76.0 8 R318Y/R338E/T343R/ R170E/T175R/R233E/ R403E/E410N E240NY155F/K228N/K247N/ Y[155]F/K63N/K82N/ 30.7 2.3 40.6 2.0 53.5 2.5 2N249S/R318Y/R338E/ N84S/R150Y/R170E/ T343R/R403E/E410N T175R/R233E/E240NY155F/R338E/T343R/ Y[155]F/R170E/T175R/ 29.8 n.d. 37.9 n.d. 50.1 n.d. 1R403E R233E R338E/T343R/R403E R170E/T175R/R233E 29.4 n.d. 37.0 n.d. 49.8n.d. 1 Y155F/R338E/T343R/ Y[155]F/R170E/T175R/ 28.3 n.d. 33.3 n.d. 44.4n.d. 1 R403E/E410S R233E/E240S Y155F/N260S/R338E/ Y[155]F/N95S/R170E/40.5 n.d. 52.9 n.d. 70.1 n.d. 1 T343R/R403E T175R/R233EY155F/I251S/R338E/T343R/ Y[155]F/I86S/R170E/ 31.9 n.d. 40.1 n.d. 54.5n.d. 1 R403E T175R/R233E R318Y/R338E/T343R/ R150Y/R170E/T175R/ 27.4 n.d.34.0 n.d. 43.3 n.d. 1 R403E/E410S R233E/E240S Y155F/K247N/N249S/Y[155]F/K82N/N84S/ 43.2 n.d. 58.6 n.d. 74.2 n.d. 1 T343R/R403ET175R/R233E Y155F/K247N/N249S/ Y[155]F/K82N/N84S/ 32.5 n.d. 41.4 n.d.55.4 n.d. 1 R318Y/R338E/T343R/ R150Y/R170E/T175R/ R403E R233EK247N/N249S/R318Y/ K82N/N84S/R150Y/ 30.8 4.2 39.1 6.9 52.5 9.1 2R338E/T343R/R403E R170E/T175R/R233E Y155F/K247N/N249S/Y[155]F/K82N/N84S/ 27.3 n.d. 34.9 n.d. 47.7 n.d. 1 R338E/T343R/R403E/R170E/T175R/R233E/ E410N E240N K247N/N249S/R338E/ K82N/N84S/R170E/ 28.2n.d. 35.1 n.d. 47.3 n.d. 1 T343R/R403E/E410N T175R/R233E/E240NY155F/K247N/N249S/ Y[155]F/K82N/N84S/ 29.6 n.d. 37.4 n.d. 48.7 n.d. 1R318Y/R338E R150Y/R170E Y155F/K247N/N249S/ Y[155]F/K82N/N84S/ 39.6 n.d.49.7 n.d. 65.0 n.d. 1 R318Y/T343R R150Y/T175R Y155F/K247N/N249S/Y[155]F/K82N/N84S/ 52.2 n.d. 67.9 n.d. 79.9 n.d. 1 R318Y/R403ER150Y/R233E Y155F/K247N/N249S/ Y[155]F/K82N/N84S/ 32.9 n.d. 43.8 n.d.55.8 n.d. 1 R318Y/E410N R150Y/E240N Y155F/K247N/N249S/Y[155]F/K82N/N84S/ 39.2 n.d. 50.4 n.d. 62.6 n.d. 1 R338E/R403ER170E/R233E Y155F/K247N/N249S/ Y[155]F/K82N/N84S/ 27.4 n.d. 31.5 n.d.41.8 n.d. 1 R338E/T343R R170E/T175R Y155F/K247N/N249S/Y[155]F/K82N/N84S/ 28.7 0.4 32.7 0.1 41.8 0.9 2 R318Y/R338E/T343R/R150Y/R170E/T175R/ E410N E240N K247N/N249S/R318Y/ K82N/N84S/R150Y/ 28.00.8 32.7 0.8 42.4 0.3 2 R338E/T343R/E410N R170E/T175R/E240NY155F/K247N/N249S/ Y[155]F/K82N/N84S/ 38.9 n.d. 50.4 n.d. 65.5 n.d. 1R318Y/T343R/R403E/ R150Y/T175R/R233E/ E410N E240N K247N/N249S/R318Y/K82N/N84S/R150Y/ 35.9 4.2 46.6 6.0 60.9 7.8 2 T343R/R403E/E410NT175R/R233E/E240N Y155F/K247N/N249S/ Y[155]F/K82N/N84S/ 27.1 1.9 31.82.0 41.2 0.8 2 R338E/E410N R170E/E240N Y155F/K247N/N249S/Y[155]F/K82N/N84S/ 44.3 n.d. 60.7 n.d. 75.5 n.d. 1 R318Y/T343R/R403ER150Y/T175R/R233E K247N/N249S/R318Y/ K82N/N84S/R150Y/ 45.3 n.d. 57.5n.d. 75.7 n.d. 1 T343R/R403E T175R/R233E Y155F/K247N/N249S/Y[155]F/K82N/N84S/ 44.9 0.1 52.5 3.7 64.9 0.5 2 R318Y/T343R/E410NR150Y/T175R/E240N K247N/N249S/R318Y/ K82N/N84S/R150Y/ 42.7 n.d. 50.2n.d. 64.6 n.d. 1 T343R/E410N T175R/E240N Y155F/K247N/N249S/Y[155]F/K82N/N84S/ 31.1 n.d. 40.9 n.d. 56.2 n.d. 1 R338E/T343R/R403ER170E/T175R/R233E K247N/N249S/R338E/ K82N/N84S/R170E/ 32.0 n.d. 43.2n.d. 56.1 n.d. 1 T343R/R403E T175R/R233E Y155F/K247N/N249S/Y[155]F/K82N/N84S/ 28.5 n.d. 32.2 n.d. 45.9 n.d. 1 R338E/T343R/E410NR170E/T175R/E240N K247N/N249S/R338E/ K82N/N84S/R170E/ 25.1 3.9 29.9 5.041.1 8.0 2 T343R/E410N T175R/E240N Y155F/K247N/N249S/ Y[155]F/K82N/N84S/36.7 n.d. 49.3 n.d. 65.4 n.d. 1 T343R/R403E/E410N T175R/R233E/E240NY155F/R318Y/R338E/ Y[155]F/R150Y/R170E/ 27.4 1.0 31.4 1.7 40.7 0.4 2T343R T175R R318Y/R338E/T343R R150Y/R170E/T175R 20.5 n.d. 24.3 n.d. 32.2n.d. 1 Y155F/R318Y/T343R/ Y[155]F/R150Y/T175R/ 43.4 n.d. 56.1 n.d. 71.3n.d. 1 R403E R233E Y155F/T343R/R403E/ Y[155]F/T175R/R233E/ 36.1 n.d.47.5 n.d. 63.0 n.d. 1 E410N E240N Y155F/K247N/N249S/ Y[155]F/K82N/N84S/28.0 1.4 32.9 0.8 42.6 0.4 2 R318Y/R338E/T343R R150Y/R170E/T175RK247N/N249S/R318Y/ K82N/N84S/R150Y/ 27.4 1.2 32.7 0.2 42.4 3.1 2R338E/T343R R170E/T175R Y155F/K247N/N249S/ Y[155]F/K82N/N84S/ 36.2 4.544.8 5.9 54.4 4.2 5 T343R/E410N T175R/E240N Y155F/K247N/N249S/Y[155]F/K82N/N84S/ 47.2 n.d. 60.7 n.d. 74.2 n.d. 1 R403E/E410NR233E/E240N Y155F/R338E/T343R/ Y[155]F/R170E/T175R/ 24.9 4.4 27.5 4.434.9 4.4 4 E410N E240N R338E/T343R/E410N R170E/T175R/E240N 19.8 n.d.23.9 n.d. 34.7 n.d. 1 Y155F/R318Y/T343R/ Y[155]F/R150Y/T175R/ 41.3 5.749.5 6.0 63.4 6.0 2 E410N E240N R318Y/T343R/E410N R150Y/T175R/E240N 34.5n.d. 44.8 n.d. 61.0 n.d. 1 K228N/R318Y/R338E/ K63N/R150Y/R170E/T175R/23.4 n.d. 28.8 n.d. 38.9 n.d. 1 T343R/R403E/E410N R233E/E240NK228N/K247N/N249S/ K63N/K82N/N84S/ 28.6 n.d. 37.3 n.d. 47.9 n.d. 1R318Y/R338E/T343R/ R150Y/R170E/T175R/ R403E R233E K228N/247N/N249S/K63N/K82N/N84S/R150Y/ 21.4 n.d. 25.8 n.d. 34.3 n.d. 1 R318Y/R338E/T343R/R170E/T175R/E240N E410N K228N/K247N/N249S/ K63N/K82N/N84S/ 35.4 n.d.44.0 n.d. 61.4 n.d. 1 R318Y/T343R/R403E/ R150Y/T175R/R233E/ E410N E240N

Since modifications will be apparent to those of skill in this art, itis intended that this invention be limited only by the scope of theappended claims.

What is claimed is:
 1. A nucleic acid molecule encoding a modifiedfactor IX (FIX) polypeptide, comprising an amino acid replacementcorresponding to T343R or T343K in an unmodified FIX polypeptide,wherein: corresponding amino acid residues are identified by alignmentof the unmodified FIX polypeptide with the polypeptide of SEQ ID NO:3;the unmodified FIX polypeptide comprises a sequence of amino acids setforth in any of SEQ ID NOS: 2, 3, 20 or 325, or a sequence of amino acidresidues having at least 95% sequence identity to the FIX polypeptidesequence set forth in any of SEQ ID NOS: 2, 3, 20 or 325, or is acatalytically active fragment thereof; the modified FIX polypeptide,when an active form, exhibits one or both of increased catalyticactivity and increased procoagulant activity compared with theunmodified FIX polypeptide; and the modified FIX polypeptide does notcontain the amino acid replacements corresponding to F342I/T343R/Y345T.2. The nucleic acid molecule of claim 1, wherein the amino acidreplacement corresponds to T343R.
 3. The nucleic acid molecule of claim1, wherein the encoded modified FIX polypeptide further comprises theamino acid replacement R338E or R338D.
 4. The nucleic acid molecule ofclaim 2, wherein the encoded modified FIX polypeptides further comprisesthe amino acid replacement corresponding to R338E.
 5. The nucleic acidmolecule polypeptide of claim 4, wherein the amino acid replacement atposition 343 corresponds to T343R, whereby the modified FIX polypeptidecomprises R338E/T343R.
 6. A nucleic acid molecule encoding a modifiedfactor IX (FIX) polypeptide, comprising the amino acid replacementsR318Y, R338E and T343, in an unmodified FIX polypeptide, wherein:corresponding amino acid residues are identified by alignment of theunmodified FIX polypeptide with the polypeptide of SEQ ID NO:3; theunmodified FIX polypeptide comprises a sequence of amino acids set forthin any of SEQ ID NOS: 2, 3, 20 or 325, or a sequence of amino acidresidues having at least 95% sequence identity to the FIX polypeptidesequence set forth in any of SEQ ID NOS: 2, 3, 20 or 325; and themodified FIX polypeptide, when an active form, exhibits one or both ofincreased catalytic activity and increased procoagulant activitycompared with the unmodified FIX polypeptide.
 7. The nucleic acidmolecule of claim 6, wherein the encoded modified FIX polypeptidecomprises an amino acid replacement at residue E410 or at an amino acidresidue corresponding to 410 in an unmodified FIX polypeptide, whereinthe replacement amino acid is N or S.
 8. The nucleic acid molecule ofclaim 7, wherein the amino acid replacement is N.
 9. The nucleic acidmolecule of claim 6, wherein the encoded modified FIX polypeptidecomprises an amino acid replacement at residue 403 of a mature FIXpolypeptide having a sequence set forth in SEQ ID NO:3 or at amino acidresidues corresponding to residue 403 in an unmodified FIX polypeptide,wherein the replacement is E.
 10. The nucleic acid molecule of claim 6,wherein the encoded FIX polypeptide comprises the amino acidreplacements R318Y, R338E, T343R, R403E and E410N in a mature FIXpolypeptide having a sequence set forth in SEQ ID NO:3 or the samereplacements at corresponding amino acid residues in the unmodified FIXpolypeptide.
 11. The nucleic acid molecule of claim 1, wherein theencoded FIX polypeptide comprises amino acid replacements selected fromamong R338E/T343R/E410N; Y155F/R338E/T343R/E410N;Y155F/K247N/N249S/T343R/E410N; Y155F/T343R/R403E/E410N;K247N/N249S/T343R/R403E/E410N; Y155F/K247N/N249S/T343R/R403E/E410N;K247N/N249S/R338E/T343R/E410N; Y155F/K247N/N249S/R338E/T343R/E410N;K247N/N249S/R338E/T343R/R403E; Y155F/K247N/N249S/R338E/T343R/R403E;Y155F/K247N/N249S/R338E/T343R; K247N/N249S/R338E/T343R/R403E/E410N;Y155F/K247N/N249S/R338E/T343R/R403E/E410N;Y155F/K247N/N249S/T343R/R403E; Y155F/I251S/R338E/T343R/R403E;Y155F/N260S/R338E/T343R/R403E; Y155F/R338E/T343R/R403E/E410S;R338E/T343R/R403E; Y155F/R338E/T343R/R403E;Y155F/R338E/T343R/R403E/E410N; R338E/T343R/R403E/E410N and R338E/T343R.12. The nucleic acid molecule of claim 1, wherein the encoded FIXpolypeptide comprises amino acid replacements selected from amongK247N/N249S/R338E/T343R/R403E/E410N, R338E/T343R/E410N and R338E/T343R.13. The nucleic acid molecule of claim 6, wherein the encoded FIXpolypeptide comprises amino acid replacements selected from amongreplacements R318Y/R338E/T343R/R403E/E410N, R318Y/R338E/T343R/E410N,Y155F/R318Y/R338E/T343R/R403E,Y155F/K228N/K247N/N249S/R318Y/R338E/T343R/R403E/E410N,Y155F/K247N/N249S/R318Y/R338E/T343R/R403E,K247N/N249S/R318Y/R338E/T343R/R403E, R318Y/R338E/T343R,Y155F/K247N/N249S/R318Y/R338E/T343R,K228N/R318Y/R338E/T343R/R403E/E410N,K228N/K247N/N249S/R318Y/R338E/T343R/R403E,R318Y/R338E/T343R/R403E/E410S, K247N/N249S/R318Y/R338E/T343R,K247N/N249S/R318Y/R338E/T343R/E410N,Y155F/K247N/N249S/R318Y/R338E/T343R/E410N,Y155F/K247N/N249S/R318Y/T343R/R403E/E410N, Y155F/R318Y/R338E/T343R andK228N/K247N/N249S/R318Y/R338E/T343R/E410N, or the same replacements atcorresponding amino acid residues in the unmodified FIX polypeptide. 14.The nucleic acid molecule of claim 6, comprising an amino acidreplacement R318Y/R338E/T343R/R403E/E410N, R318Y/R338E/T343R/E410N,Y155F/R318Y/R338E/T343R/R403E,Y155F/K228N/K247N/N249S/R318Y/R338E/T343R/R403E/E410N,Y155F/K247N/N249S/R318Y/R338E/T343R/R403E,K247N/N249S/R318Y/R338E/T343R/R403E, R318Y/R338E/T343R,Y155F/K247N/N249S/R318Y/R338E/T343R,K228N/R318Y/R338E/T343R/R403E/E410N,K228N/K247N/N249S/R318Y/R338E/T343R/R403E, R318Y/R338E/T343R/R403E/E410Sor K247N/N249S/R318Y/R338E/T343R.
 15. The modified FIX polypeptide ofclaim 6, comprising amino acid replacements R318Y/R338E/T343R,R318Y/R338E/T343R/R403E/E410N or R318Y/R338E/T343R/E410N.
 16. Themodified FIX polypeptide of claim 6, further comprising the amino acidreplacement Y155F or the same replacement at a corresponding amino acidresidue in the unmodified FIX polypeptide.
 17. A nucleic acid molecule,wherein: the encoded modified FIX polypeptide comprises the sequence ofamino acids set forth in any of SEQ ID NOS: 267, 330, 331, 334, 336,345-348, 353, 354, 357-360, 366, 368, 369, 378, 379, 393, 394, 397, 398and 405-407; or the encoded modified FIX polypeptide comprises asequence of amino acids that exhibits at least 95% amino acid sequenceidentity with the sequence of amino acids set forth in any of SEQ IDNOS: 267, 330, 331, 334, 336, 345-348, 353, 354, 357-360, 366, 368, 369,378, 379, 393, 394, 397, 398 and 405-407 and comprises the aminoreplacements corresponding to R318Y, R338E and T343R, wherein:corresponding replacements are identified by alignment with SEQ ID NO:3;and, the modified FIX polypeptide, when activated, exhibits one or bothof increased catalytic activity and increased procoagulant activitycompared with the unmodified FIX polypeptide lacking the R318Y, R338Eand T343R replacements.
 18. The nucleic acid molecule of claim 1,wherein the unmodified FIX polypeptide consists of a sequence of aminoacids set forth in any of SEQ ID NOS: 2, 3, 20 or
 325. 19. The nucleicacid molecule of claim 6, wherein the unmodified FIX polypeptideconsists of a sequence of amino acids set forth in any of SEQ ID NOS: 2,3, 20 or
 325. 20. The nucleic acid molecule of claim 1 wherein theencoded FIX polypeptide comprises the sequence of amino acids set forthin SEQ ID NO:394.
 21. The nucleic acid molecule of claim 1 wherein theencoded FIX polypeptide is FIXa polypeptide.
 22. A vector, comprisingthe nucleic acid molecule of claim
 1. 23. A vector, comprising thenucleic acid molecule of claim
 6. 24. A pharmaceutical composition,comprising the nucleic acid molecule of claim
 1. 25. A pharmaceuticalcomposition, comprising the nucleic acid molecule of claim
 6. 26. Apharmaceutical composition, comprising the vector of claim
 22. 27. Apharmaceutical composition, comprising the vector of claim
 23. 28. Anon-human cell or a cell culture, comprising the nucleic acid moleculeof claim
 1. 29. A non-human cell or a cell culture, comprising thenucleic acid molecule of claim
 6. 30. A T-cell, comprising the nucleicacid molecule of claim
 1. 31. A T-cell, comprising the nucleic acidmolecule of claim
 6. 32. A method, comprising administering a nucleicacid molecule of claim 1 to a subject, wherein the disease or conditionto be treated is selected from among blood coagulation disorders,hematologic disorders, hemorrhagic disorders, hemophilias, and bleedingdisorders.
 33. A method, comprising administering a nucleic acidmolecule of claim 6 to a subject, wherein the disease or condition to betreated is selected from among blood coagulation disorders, hematologicdisorders, hemorrhagic disorders, hemophilias, and bleeding disorders.34. The method of claim 32, wherein the hemophilia is hemophilia B. 35.The method of claim 33, wherein the hemophilia is hemophilia B.